Reperfusion with omega-3 glycerides promotes donor organ protection for transplantation

ABSTRACT

It has been discovered that isolated organs and tissues perfused/reperfused in perfusion buffer to which omega-3 glyceride oil had been added retain higher levels of function than if perfused/reperfused without the omega-3 glycerides. Isolated hearts reperfused ex vivo after induced ischemia in n-3 triglyceride perfusion emulsion maintained a normal heart rate and normal LVDP and showed a dramatically reduced frequency of arrhythmias compared to control hearts. Further, test hearts reperfused with n-3 oil triglyceride emulsion showed a decrease in creatine kinase and upregulation of certain beneficial proteins including the anti-apoptotic gene marker Bcl-2.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 14/780,832 filed Sep. 28, 2015 which is a 371 national stageapplication of PCT Application No. PCT/US14/032279, filed Mar. 28, 2014,and claims the benefit of U.S. Provisional Application No. 61/806,391,filed on Mar. 28, 2013; the entire contents of which are herebyincorporated by reference as if fully set forth herein.

STATEMENT OF GOVERNMENTAL INTEREST

This invention was made with government support under grant HL040404awarded by the National Institute of Health. The Government has certainrights in this invention.

BACKGROUND

The present invention provides preservation solutions useful for storingorgans while awaiting implantation which extend the vitality of theorgan and reduce damage to organ cells. The present invention alsoprovides method for preserving organs which extend the maximum life ofthe organ before and during transplantation.

A great deal of research progress has been made over the years inunderstanding cellular mechanisms, as well as developing newtransplantation techniques, for keeping organs viable not only duringstorage but also after reperfusion of these organs (e.g., minimizationof ischemia/reperfusion injury). As a result, organ transplantation hasbecome an established and effective technique. A significant factorlimiting clinical application of organ transplantation is decrease inviability of the organ after removal from the donor.

Generally, the two most frequently used methods for preserving organsafter removal from the donor are simple hypothermic storage andcontinuous pulsatile perfusion. With simple hypothermic storage, theorgan is removed from the donor and cooled rapidly. This is usuallyachieved by a combination of cooling and short periods of perfusion todrop the organ temperature as quickly as possible to a temperaturebetween 0° C. and 4° C., where it may be held for up to about six hours.While cold storage enables organs to be transplanted, the time duringwhich the organ is viable is short. Cold storage decreases the rate atwhich intracellular enzymes, essential cellular components necessary fororgan viability, degrade but does not stop metabolism entirely.

The second method of organ preservation which has undergone extensiveinvestigation, continuous pulsatile perfusion, utilizes the followingelements: (1) pulsatile flow, (2) hypothermia, (3) membrane oxygenation,and (4) a perfusate containing both albumin and lipids. Although beingmore technically complex and costly, continuous pulsatile perfusionprovides significantly longer viability of the organ when compared tosimple hypothermia.

Preserving organs at between 0° C. and 4° C. can result in damage to theorgan during storage and upon reperfusion with a warm reperfusionsolution. Injury to the organ occurs through damage to epithelial,endothelial and parenchymal cells. Although some of the solutions of theprior art have been useful to extend the storage time of donor organsand lessen injury to the organ upon reperfusion, cell injury still doesoccur frequently. It is desirable to extend the viable organ life andimprove the quality of the transplanted organ. For example, usingpreservation solutions of the prior art, kidneys that have been in coldstorage beyond 48 hours frequently cannot be used and must be discarded.Extending organ viability allows sufficient time for compatibilitytesting of the donor and recipient, and increased organ availability. Itis also desirable to minimize damage to the organ upon reperfusion.Ischemia-reperfusion injury to transplanted organs preserved insolutions of the prior art commonly results in delayed graft function,and predisposes the graft to acute and chronic rejection.

There remains a need in the art for improved methods and compositionsfor conservation and preparation of organs and tissues for transplant.

SUMMARY

Certain embodiments are directed to omega-3 oil in water glycerideemulsions for perfusion of an organ or tissue ex vivo, wherein theemulsion comprises (a) a perfusion buffer suitable for organ or tissuepreservation and transplantation, (b) less than 7% (preferably less than2%) of an omega-3 oil by weight in grams per 100 ml of perfusion buffer,wherein the omega-3 oil (i) comprises from about 10% to about 99%omega-3 diglyceride, omega-3 triglyceride or combinations thereof byweight per total weight of the omega-3 oil, and at least about 20% toabout 99% of the total acyl groups of the omega-3 diglycerides andtriglycerides comprise EPA or DHA, and (ii) comprises less than 10%omega-6 fatty acids by weight per total weight of the omega-3 oil. Theemulsion itself has (c) less than 10% omega-6 oil, and (d) the meandiameter of lipid droplets in the emulsion is from about 100 nm to lessthan about 5 microns, preferably from 100 nm to 500 nm.

Other embodiments are directed to methods of preserving isolated organsand tissues, comprising (a) providing the omega-3 oil in water emulsionof the invention, and (b) contacting (including perfusing, storing, andreperfusing) the isolated organ or tissue ex vivo with the emulsion fora duration of time at a temperature that preserves the organ or tissueuntil it is transplanted into a recipient or is otherwise used. Organsand tissues for use in the present methods perfusing, storing, andreperfusing) include liver, lung, skin, heart, heart valve, bone, bonemarrow, blood vessels, lymph nodes, intestine, pancreas, teeth, gingiva,small bowel, colon, appendages (fingers, toes, arms, legs), scalp,epithelium or blood for transfusion.

Other embodiments are directed to methods for preserving or preparing anorgan or tissue for transplantation or other use, comprising contacting(including perfusing, storing, and reperfusing) the organ or tissue exvivo with the omega-3 emulsion of the invention for a duration of timeat a temperature that preserves the organ or tissue until it istransplanted into a recipient or is otherwise used. Storage andperfusion/reperfusion temperatures can vary widely, typically from about0° C. to about 37° C. In some embodiments the organ or tissue iscontacted with the omega-3 glyceride perfusion emulsions of theinvention by static cold storage or low temperature continuousperfusion/reperfusion, at a temperature within in a range of about −5°C. to about 10° C.

In some embodiments n-3 glyceride emulsions are added to storagesolutions, and certain embodiments are directed to storage solutionscomprising the emulsions. Omega-3 emulsions may also be added tomatrices that may be used to preserve some tissues before transplantsuch as skin, and some embodiments are directed to these matricescomprising n-3 glyceride emulsions. Any perfusion buffer, storagesolution or matrix used for preserving, storing, perfusing organs ortissues can be used in the embodiments, typically but not necessarilyalways at a physiologic pH.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way of example, and not by wayof limitation, in the following figures.

FIG. 1 is an illustration that shows the ex vivo Langendorff system(equipment and apparatus) used to study isolated C57BL/6 mouse heartsperfused immediately after isolation with a perfusion buffer containingKREBS solution with glucose. After a 30 min. period of ischemia, theperfused hearts were then reperfused with the same perfusion buffer with(test reperfusion emulsion) or without (control reperfusion buffer) anomega-3 (n-3) triglyceride (TG). In the example, the amount(concentration) of n-3 TG in the test reperfusion buffer was 300 mg n-3TG/100 ml (final concentration of 0.3%) and 48% of the n-3 TG fattyacids (FA) were EPA and DHA.

FIG. 2 is an illustration that includes graphs that show the effects ofreperfusion with the test n-3 TG emulsion on left ventricular developedpressure (LVDP) after ischemia.

FIG. 3 is a bar graph that illustrates LVDP recovery vs. baselinefurther showing that test reperfusion emulsion with n-3 TG resulted inalmost 100% recovery vs. control reperfusion buffer without n-3 TG˜40%.

FIG. 4 is a bar graph that illustrates an increase in % heart rate aftern-3 TG emulsion administration.

FIG. 5 is a bar graph that illustrates infusion of an n-6 triglycerideemulsion Intralipid,™ decreased recovery after ischemia from 40% to only22%. This suggests that n-6 glycerides may actually have adversr effectson cardiac tissue recovery after ischemia.

FIG. 6 is a bar graph that illustrates n-3 TG markedly decreasedcreatine kinase levels in the perfusion solution, a measure of hearttissue injury, after ischemia.

FIG. 7A-7B are graphs that illustrate Bcl-2, an anti-apoptotic marker,mildly increases after ischemia in controls, but n-3 TG added to thereperfusion buffer increased it much more. FIG. 7A is a bar graph thatshows mRNA gene expression and FIG. 7B is a photograph that shows theactual protein measured by western blot.

FIG. 8A-8C are bar graphs that illustrate HIF1, associated withdecreasing apoptosis and decreasing cell death, was down-regulated afterreperfusion in n-3 TG emulsion as is shown both by gene expression inbar graphs (FIGS. 8A-8B) and by western blot analysis for HIF1 proteinas shown in FIG. 8C (bottom left for western gel, bottom right forprotein).

FIG. 9A-9C show that the autophagic marker Beclin-1 was decreased in exvivo hearts after ischemia when n-3 TG was added to the reperfusionbuffer. FIG. 9A is a bar graph that shows the level of Beclin-2 mRNAexpression after H/I in control hearts and hearts reperfused after H/Iwith n-3 glyceride perfusion emulsion. FIG. 9B is a photograph thatshows western blot analysis for Beclin-1 protein (bottom left forwestern gel, bottom right for protein). FIG. 9C is a bar graph thatshows the level of Beclin-1 protein.

FIG. 10A-10B show that PPARγ protein expression increase after I/R(ischemia after the initial baseline perfusion followed by reperfusionin Control buffer) but decreased by inclusion of n-3 TG in thereperfusate as shown in the bar graph represented by FIG. 10A. FIG. 10Bis a photograph showing western blot analysis for PPAR gamma protein.

FIG. 11A-11C show the effects on hearts in vivo of acute n-3 TGemulsions administered after H/I in the in vivo left anterior descendingcoronary artery (LAD) occlusion model. Mice were subjected to LADocclusion for 30 min followed by reperfusion period (48 h) in vivo withor without acute n-3 TG emulsion injection. FIG. 11A is a bar graph thatshows that hearts isolated at 48 h of reperfusion and subjected to TTCstaining n=3 mice/group. *P<0.05. FIG. 11B is a bar graph that showstotal plasma LDH levels at 48 hrs. n=3 mice/group. *P<0.05. FIG. 11C isa bar graph that quantifies fractional shortening percentage detected byechocardiogram just before sacrifice at 48 hrs. n=3 mice/group. *P<0.05.Data represent means±SD.

FIG. 12A-12B shows the effect of n-3 TG emulsion on an ex vivoischemia/reperfusion model. FIG. 12A is a bar graph that showsmyocardial ischemic injury by measuring left ventricular developedpressure (LVDP) recovery in hearts subjected to ischemia/reperfusiontreated with or without n-3 TG emulsion during reperfusion. *P<0.05.FIG. 12B is a bar graph that shows the level of LDH release duringreperfusion time. *P<0.05. Four hearts per group were studied. Datarepresent means±SD.

FIG. 13A-13C illustrate signaling pathways. FIG. 13A is a photographthat shows western blot analysis of p-AKT and p-GSK3β in heartssubjected to ischemia/reperfusion injury with or without n-3 TG emulsiontreatment. FIG. 13B is a bar graph that shows that p-AKT was increasedby n-3 TG treatment. FIG. 13C is a bar graph that shows that GSK3b wereincreased by n-3 TG treatment. *P<0.05. Three hearts per group werestudied. Data represent means±SD.

FIG. 14A-14C illustrate markers for apoptosis and autophagy. FIG. 14A isa photograph that shows western blot analysis of Bcl-2 and Beclin-1 inhearts subjected to I/R injury with or without n-3 TG emulsionadministered during reperfusion time. FIG. 14B is a bar graph that showsincreased: Bcl-2 and FIG. 14C is a bar graph that shows decreasedBeclin-1 protein expression in n-3 TG treated hearts. *P<0.05. Threehearts per group were studied. Data represent means±SD.

FIG. 15A-15C illustrate examples of transcription factors expression.FIG. 15A is a photograph that shows western blot analysis of PPAR-γ andHIF-1a in hearts subjected to I/R injury with or without n-3 TG emulsionadministered during reperfusion time. FIG. 15B is a bar graph that showsHIF-1a. FIG. 15C is a bar graph that shows PPAR-γ expression which weredecreased by n-3 TG administration. *P<0.05. Three hearts per group werestudied. Data represent means±SD.

FIG. 16A-16B are bar graphs that show release of the injury marker LDH.FIG. 16A is a bar graph that shows LDH release (fold change) compared toI/R control (reported in FIG. 12A-12B). p-AKT inhibitor (10 μMLY-294002) increased LDH. Treatment with GSK3β inhibitor (3 μM SB216763)significantly inhibited LDH release. P<0.05. Data represent means±SD.FIG. 16B is a bar graph that illustrates LDH release (fold change)compared to n-3 TG I/R (reported in FIG. 12A-12B). p-AKT inhibitorabolished the beneficial effect of n-3 TG. GSK3β inhibitor (3 μMSB216763). Rosiglitazone treatment entirely reversed the protectiveeffect of n-3 TG. P<0.05. Six hearts per group were studied. Datarepresent means±SD. *P<0.05 I/R vs treatments.

FIG. 17 illustrates plasma glucose concentrations (mg/dL) in non-fastingmice (p10) in post-H/I treatment of n-3 TG or n-6 TG or vehicle (saline)comparing to the time between before H/I and after H/I. **p<0.001 (n=5-9in each group).

FIG. 18A-18C illustrate TTC stained coronal sections of mouse brain andquantification of injury after H/I. FIG. 18A is a micrograph thatillustrates TTC-stained coronal sections of representative mouse brainsfrom saline treated, n-3 TG treated and n-6 TG treated. The top panelshows images of coronal mouse brain sliced and stained with TTC (greyfor living tissue and white for the infarcted tissue), and the lowerpanel shows the infarcted areas traced in black for quantification. FIG.18B is a bar graph that illustrates percent of cerebral infarct volumefrom pre-H/I mice treated with n-3 TG emulsion (n=28) or n-6 TG emulsion(n=10) or saline control (n=27). FIG. 18C is a bar graph thatillustrates percent of cerebral infarct volume after H/I in the post-H/Itreatment protocol in mice treated with n-3 TG emulsion (n=18) or salinecontrol (n=18). Each bar represents the mean±SEM of 5-7 independentexperiments.

FIG. 19A-19B illustrate the effect of Tri-DHA versus Tri-EPA on cerebralinfarct volume after H/I. FIG. 19A illustrates mice subjected to 15 minischemia followed by 24-hr reperfusion and received 2 i.p.administrations (immediately after ischemia and 1 hr of reperfusion) at2 doses (0.1 g n-3 TG/kg and 0.375 g n-3 TG/kg). Each bar represents themean±SEM of 5-7 independent experiments performed using the same H/Imodel. FIG. 19B are micrographs that illustrate TTC-stained coronalsections of representative mouse brains from saline treated, 0.1 gTri-DHA, 0.375 g Tri-DHA, 0.1 g Tri-EPA and 0.375 g Tri-EPA. *p<0.05compared to other groups except 0.1 g Tri-DHA/kg. **p<0.05 compared toother groups except 0.375 g Tri-DHA/kg and 0.375 g Tri-EPA/kg.

FIG. 20 is a bar graph that illustrates the effects of delayed treatmentwith Tri-DHA on cerebral infarct volume after H/I. Mice were subjectedto 15-min ischemia followed by 24-hr reperfusion and received 2 i.p.administrations at four-time points (immediate [0,1 hr], delayed 1-hr[1,2 hr], or 2-hr [2,3 hr] or 4-hr [4,5 hr] treatments). Each barrepresents the mean±SEM of 5-7 independent experiments. *p<0.05;**p<0.001 vs. saline control (n=10-20 in each group).

FIG. 21 illustrates the long-term effect of Tri-DHA on cerebral tissuedeath at 8 wk after H/I. Mice were subjected to 15-min H/I and received2 i.p. administrations of 0.375 g Tri-DHA/kg (n=6) vs. saline (n=5).Right brain tissue loss in relation to the contralateral hemisphere wascalculated and expressed as a percentage. Each bar represents themean±SEM. *p<0.05.

FIG. 22A-22B illustrate acute n-3 TG injection which decreased brainCa²⁺ induced opening of mitochondrial permeability transition pores(mPTP) after H/I. After H/I and after n-3 TG injection, mitochondrialfunction is maintained.

FIG. 23A-23C are bar graphs that illustrate the reduction of acute MI invivo after administration of n-3 TG. FIG. 23A is a bar graph thatillustrates the reduction of infarct size area (%) for (left) controland (right) after administration of n-3 TG in the mouse heart after H/Iinjury. FIG. 23B is a bar graph that illustrates a decrease in LDHrelease which is a marker for heart cell damage for (left) control and(right) after administration of n-3 TG. FIG. 23C is a bar graph thatillustrates n-3 TG maintenance of heart function via fractionalshortening (%) for (left) control and (right) after administration ofn-3 TG. Each bar represents the mean±SEM. *p<0.01.

FIG. 24 are LVDP graphs of marked increases of arrhythmias afterischemia observed in the Langendorff system by pressure recording incontrol hearts perfused and reperfused with their “basic” buffer.Results are shown for 2 different representative hearts.

FIG. 25 are LVDP graphs in the Langendorff system for n-3 TG treatedhearts showing a normal heart rate, normal LVDP, and very fewarrhythmias compared to the control hearts in FIG. 24. Results are shownfor 2 different representative hearts.

Before the present embodiments of the invention are described, it is tobe understood that the inventions are not limited to the particularprocesses, compositions, or methodologies described, as these may vary.The terminology used in the description is for the purpose of describingthe particular versions or embodiments only, and is not intended tolimit the scope of the present invention. Unless otherwise defined, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art. Although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of embodiments of the presentinvention, the preferred methods, devices, and materials are nowdescribed. All publications mentioned herein, are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that the invention is not entitled to antedate such disclosureby virtue of prior invention.

DETAILED DESCRIPTION

In the following description, for the purposes of explanation, numerousspecific details are set forth in order to provide a thoroughunderstanding of the present invention. It will be apparent, however, toone skilled in the art that the present invention may be practicedwithout these specific details.

1. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics, protein, and nucleic acid chemistry and hybridizationdescribed herein are those well-known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates (1992, and Supplements to 2002); Harlow andLan, Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1990); Principles of Neural Science,4th ed., Eric R. Kandel, James H. Schwart, Thomas M. Jessell editors.McGraw-Hill/Appleton & Lange: New York, N. (2000).

As used herein “omega-3 di- or triglyceride oil” means an omega-3 oilcomprising di- or triglycerides or combinations thereof, that have lessthan 10% omega-6 oil, preferably less than 5% omega-6 oil. As usedherein, “omega-3 oils” means any omega-3 fatty acid, including freeomega-3 fatty acids and omega-3 triglycerides, diglycerides andmonoglycerides.

As used herein “omega-3 (n-3) oil-in-water perfusion emulsions” and “n-3glyceride perfusion emulsions” are used interchangeably to meann-3-containing emulsions for use as perfusion buffers for storing,perfusing, reperfusing or otherwise contacting an isolated organ ortissue, for example one intended for transplantation. The n-3 glycerideperfusion emulsions of the invention have less than 7% of an omega-3 oiland less than 10% omega-6 oil by weight in grams per 100 ml of buffer.The omega-3 oil in the emulsion comprises at least 20% omega-3triglyceride (n-3 TG), omega-3 diglyceride (n-3 DG) or combinationsthereof by weight per total weight of the omega-3 oil; less than about10% omega-6 (n-6) fatty acids; and at least about 20% to about 100% ofthe total acyl groups of the omega-3 of the diglycerides ortriglycerides consist of EPA and/or DHA.

As used herein, “omega-3 fatty acid” means a polyunsaturated fatty acidwherein one of the carbon-carbon double bonds is between the third andfourth carbon atoms from the distal end of the hydrocarbon side chain ofthe fatty acid. Examples of “omega-3 fatty acid” include α-linolenicacid (18:3n-3; α-ALA; Δ^(3,6,9)), eicosapentaenoic acid (20:5n-3; EPA;Δ^(5,8,11,14,17)), docosahexaenoic acid (22:6n-3; DHA) anddocosapentaenoic acid (22:5n-3; DPA; Δ^(7,10,13,16,19)), wherein EPA andDHA are most preferred. Omega-3 fatty acids having at least 20 carbonatoms are herein called “long chain omega-3 fatty acids.”

As used herein, “omega-3 triglyceride” or “omega-3 diglyceride” or“omega-3 monoglyceride” refers to a triglyceride or a diglyceride ormonoglyceride, respectively, comprising at least one omega-3 fatty acidesterified with a glycerol moiety. As used herein, the term “omega-3tri/diglyceride” means that omega-3 fatty acid comprises an omega-3triglyceride and/or a diglyceride or any combination thereof.

As used herein, the amount of omega-3 oil (or omega-6 oil) in thelipid-based oil-in-water emulsion is expressed by weight in grams ofomega-3 (or omega-6 oil) per 100 mL emulsion.

As used herein, the amount of glyceride (mono-, di-, or triglyceride) inthe omega-3 oil (or omega-6 oil) oil is expressed as the percentage ofthe glyceride by weight per total weight of the omega-3 (or omega-6oil).

As used herein, the amount of fatty acid such as EPA or DHA in aglyceride (mono-, di-, or triglyceride) is expressed as the % of thetotal acyl groups of the respective glyceride.

As used herein, hypoxia refers to a shortage of oxygen in the body or ina specific organ or tissue.

As used herein, ischemia refers to insufficient blood flow to provideadequate oxygenation. The most common causes of ischemia are acutearterial thrombus formation, chronic narrowing (stenosis) of a supplyartery that is often caused by atherosclerotic disease, and arterialvasospasm. As blood flow is reduced to an organ, oxygen extractionincreases. When the tissue is unable to extract adequate oxygen, thepartial pressure of oxygen within the +tissue fails (hypoxia) leading toa reduction in mitochondrial respiration and oxidative metabolism.Further, in many acute situations of organ ischemia-hypoxia (e.g.,stroke, myocardial infarction, intestinal volvulus, etc.) the patient isfar too ill to have oral or enteral administration of therapeutic agentsand thus needs parenteral injections, such as from lipid emulsions forimmediate action.

As used herein, hypoxia-ischemia refers to the occurrence of bothhypoxia and ischemia in a tissue or organ.

As used herein “perfusion buffer” refers collectively to washout,preservation, intracellular and flush solutions devised and evaluatedfor cold storage of an isolated organ or tissue that has been removedfrom the body that is intended for transplantation. The isolatedorgan/tissue is perfused, reperfused, stored or otherwise contactedimmediately after removal from the body with such a buffer or solution.The use of the term “intracellular” solutions is due to theirresemblance, in some respects, to intracellular fluid.

As used herein, “perfusion” refers to perfusing an isolated organ ortissue after isolation from the body, and “reperfusion” refers tosubsequent perfusions after the organ or tissue has undergone anischemic event.

As used herein, “n-3 glyceride perfusion emulsions” of the presentinvention refer to perfusion buffers that comprise less than 7% n-3 oilby weight per 100 ml of emulsion, and less than 10% n-6 oil by weightper 100 ml of emulsion.

As used herein, the amount of n-3 oil (diglyceride, triglyceride) in theemulsion is expressed as % TG or DG by weight per total weight of then-3 oil.

As used herein, “tissue” is used herein to mean any controlled medicalsupport product or biological substance such tissues, biologicalspecimens or other medical products that require special conditionsduring transport.

As used herein, an “organ” is a collection of cells and tissues joinedin a structural unit to serve a common function. Organs include anyorgan that can be transplanted including the heart, kidneys, liver,lungs, pancreas, intestine, and thymus. Tissues include bones, tendons(both referred to as musculoskeletal grafts), composite tissueallografts, cornea, skin, heart valves, nerves and veins. A “transplantorgan” as used herein may refer to an organ that is also a donor organand/or an organ that is intended for transplantation. “Transplant organ”may refer to a donor organ that has yet to be transferred to arecipient.

As used herein, “reperfusion damage” or “reperfusion injury” are usedinterchangeably herein to refer to damage caused with restoration ofblood supply to hypoxic-ischemic (H/I) tissues either in vivo after anischemic event or ex vivo in isolated organs and tissues. Anischemia-reperfusion injury can be caused, for example, by a naturalevent (e.g., restoration of blood flow following a myocardialinfarction), a trauma, or by one or more surgical procedures or othertherapeutic interventions that restore blood flow to a tissue or organthat has been subjected to a diminished supply of blood. Such surgicalprocedures include, for example, coronary artery bypass graft surgery,coronary angioplasty, organ transplant surgery and the like.

2. SUMMARY OF EMBODIMENTS

It has been discovered that isolated hearts reperfused for an hour exvivo after 30 min. of induced ischemia, retained dramatically higherfunction when reperfused in buffer to which omega-3 triglyceride oil hadbeen added. The isolated hearts were reperfused with a dilute n-3glyceride perfusion emulsion having 0.3% n-3 triglyceride oil ingrams/100 ml of perfusion buffer. Hearts reperfused in the n-3triglyceride emulsion maintained a normal heart rate and normal leftventricular developed pressure (LVDP) and showed a dramatically reducedfrequency of arrhythmias compared to control hearts. Further, testhearts reperfused n-3 oil triglyceride emulsion showed a decrease increatine kinase and upregulation of certain beneficial proteinsincluding the anti-apoptotic gene marker Bcl-2. Reperfusion with the n-3triglyceride perfusion emulsion also down-regulated harmful proteinsincluding HIF1 (a marker for autophagy which may increase cell death),PPAR gamma (associated with some pro-inflammatory responses in heart),and Beclin 1 (an autophagic marker). These ex vivo results using a 0.3%n-3-TG-containing emulsion to reperfuse isolated hearts after inducedischemia are consistent with observations presented in Examples 3-5showing that in vivo administration of various n-3 triglyceride (TG) anddiglyceride- (DG) oil-in-water emulsions to animals having sufferedhypoxia/ischemia dramatically reduced reperfusion injury the brain andheart.

While not wishing to be bound by theory, it is believed that the omega-3fatty acid content of cell membranes of certain isolated organs (such asthe heart, lungs and brain) and tissues can be preserved and alsoincreased by being stored, perfused, reperfused or otherwise contactedwith omega-3 di- and tri-glyceride emulsions. It has been speculatedthat n-3 diglycerides are hydrolyzed faster than triglycerides, and that1, 2-diglycerides are also highly bioactive molecules. It has beenspeculated that diglyceride emulsion droplets are more efficiently takenup by organs than triglyceride emulsion droplets.

Based on the collective results described herein, certain embodiments ofthe invention are directed to new omega-3 oil-in-water perfusionglyceride emulsions (herein also “n-3 glyceride (TG or DG) perfusionemulsions”) that comprise less than 7% n-3 TG- or DG-oil by weight ingrams per 100 ml of emulsion, wherein the n-3 oil is mixed withperfusion buffer. Any standard perfusion buffer for preserving, storingand perfusing/reperfusing isolated organs and tissues ex vivo can beused in the n-3 glyceride perfusion emulsions of the invention. Theperfusion buffers are based on a balanced isotonic solution thattypically includes sodium, potassium, calcium and magnesium ions, aswell as glucose and sodium bicarbonate, in a physiologically acceptableamount. Certain of these types of solutions are well known in the art.The omega-3 glyceride perfusion emulsions of the invention are nontoxicand stable for extended periods of time.

Certain other embodiments are directed to methods of preserving anisolated organ or tissue by contacting it with an n-3 glycerideperfusion emulsion of the invention ex vivo, for example for apredetermined duration of time (typically 1 to 24 hours) at apredetermined temperature. Storage and perfusion/reperfusiontemperatures can vary widely, typically from about 0° C. to about 37° C.In some embodiments the organ or tissue is contacted with the omega-3glyceride perfusion emulsions of the invention by static cold storage orlow temperature continuous perfusion/reperfusion, at a temperaturewithin in a range of about −5° C. to about 10° C.

In an embodiment, the new n-3 glyceride perfusion emulsions comprise:

-   -   (a) a perfusion buffer suitable for organ or tissue preservation        and transplantation,    -   (b) less than 7% of an omega-3 oil by weight in grams per 100 ml        of perfusion buffer, wherein the omega-3 oil        -   (i) comprises from about 10% to about 99% omega-3            diglyceride, omega-3 triglyceride or combinations thereof by            weight per total weight of the omega-3 oil, wherein about            20% to about 99% of the total acyl groups of the            diglycerides or triglycerides consist of EPA or DHA, and        -   (ii) comprises less than 10% omega-6 fatty acids by weight            per total weight of the omega-3 oil,    -   (c) less than 10% of an omega-6 oil, and    -   (d) the mean diameter of lipid droplets in the emulsion is less        than about 5 microns.

The omega-3 glyceride perfusion emulsions of the invention can have:

-   -   (a) from about 0.5 to about 1%, 1-3%, 3-5% or 5-less than 7%        omega-3 oil;    -   (b) from about 10% to about 20%, from 20% to about 40%, from 40%        to about 60% and from 60-99% of the n-3 oil can be TG or DG; and    -   (c) from about 20% to 50%, from 50% to 75%, from 75% to 90%,        from 90% to 95% or from 95% to 100% of the total acyl groups of        the omega-3 diglycerides or triglycerides consisting of EPA or        DHA.

In some embodiments the mean diameter of lipid droplets in the emulsionis less than about 5 microns, less than 1 micron or less than 500 nm.

In certain embodiments, the omega-3 oil is fish oil, synthetic omega-3oil or a combination thereof. Other embodiments of new perfusionemulsions are described below in more detail. In certain embodiments,the omega-3 oil is about 10% to about 97% omega-3 TG and at least 30%,40% or 50% of the total acyl groups on the TG are DHA (herein “Tri-DHAperfusion emulsions”); however, this can vary widely, up to 99%. Aspecific example is the “TG90-DHA30” Tri-DHA emulsion that wasadministered in vivo following H/I to reduce reperfusion injury at aconcentration of 10% omega-3 oil by weight in grams per 100 ml ofemulsion, wherein the omega-3 oil is >90% triglyceride (TG) by weightper total weight of the omega-3 oil, and in which up to about 30% of thetotal acyl groups are DHA. In other embodiments the omega-3 oil is atleast 20%, 40%, 60% DG and at least 30%, 40% or 50% of the total acylgroups on the TG are DHA (herein “Tri-DHA perfusion emulsions”);however, this can vary widely, up to 99%.

The omega-3 TG- and DG-perfusion emulsions of the present invention arenot naturally occurring. For example, there is no omega-3 oil found innature that comprises at least 20% omega-3 diglycerides wherein at least20% of the total acyl-groups of the omega-3 di- or triglycerides consistof EPA or DHA. The highest percentage EPA plus DHA in an omega-3 oil isfound in sardine and anchovy oil, where the combined percentage EPA andDHA might be as high as only 27% to 28%. Racine R A, Deckelbaum R J.,Curr Opin Clin Nutr Metab Care. 2007 March; 10(2):123-8. Extraordinarymeasures have to be taken to obtain a triglyceride (TG) or a diglyceride(DG) n-3 oil in which more than 20% of the total acyl groups are eitherEPA or DHA, such as hydrolyzing the fatty acids off of fish oil n-3triglycerides. The DHA and EPA are then purified and separated fromother fatty acids in the preparation, and then the DHA and/or EPA arere-esterified back onto a glycerol molecule to make an n-3 DG or TGwherein more than 20% of the fatty acids are DHA and EPA.

Sources of omega-3 fatty acids may be from any suitable source such asfrom fish oils, other oils or may be synthesized. Although EPA and DHAare preferred omega-3 fatty acids, other omega-3 fatty acids may be usedsuch as docosapentaenoic acid. Suitable exemplary fish oils for use inthe present perfusion/reperfusion emulsions include oils from cold-waterfish such as salmon, sardine, mackerel, herring, anchovy, smelt andswordfish. Fish oils generally contain glycerides of fatty acids withchain lengths of 12 to 22 carbons. Non-naturally occurring, highlypurified fish oil concentrates obtained, for example, from sardine,salmon, herring and/or mackerel oils may have an eicosapentaenoic acid(EPA) content of from about 20 to 40 wt.-%, and a docosahexaenoic acid(DHA) content of >10% based on the fatty acid methyl esters of the fishoil concentrate as determined by gas chromatography (percent by area).U.S. Pat. No. 6,159,523 discloses a method for making fish oilconcentrates. Generally, the amount of the polyunsaturated fatty acidsof the omega-6 series (such as linoleic acid) in natural fish oils islow, i.e. less than 10%, and typically less than 5%. In the describedn-3 perfusion/reperfusion emulsions of the present inventions, theamount of omega-6 oil in the overall emulsions is less than 10% byweight in grams per 100 ml of perfusion buffer, wherein the omega-3 oil,preferably less than 5%; and the amount of omega-6 oil in the omega-3oil of the emulsion is also less than 10% by weight in grams per 100 mlof perfusion buffer, wherein the omega-3 oil, preferably less than 5%.N-3 TG with more than about 25% of the total acyl groups of the TGconsisting of DHA or EPA is typically synthesized and/or purified.

3. BACKGROUND

Much of the injury to transplanted organs occurs not during ischemia,but during reperfusion. This finding has led to many advances in organpreservation aimed at preventing this type of injury. Furthermore, someof the events that occur during reperfusion may result in enhancedimmunogenicity of the graft. Oxygen free-radicals generated duringreperfusion are a major cause of the reperfusion injury, but cytokinesand nitric oxide also play a role.

Damage to organs during transplantation typically occurs in two phases.The first, the warm ischemic phase, includes the time from theinterruption of circulation to the donor organ to the time the organ isflushed with hypothermic preservation solution. In multiorgan recovery,the organs are cooled before they are removed. The second, the coldischemic phase, occurs when the organ is preserved in a hypothermicstate prior to transplantation into the recipient. The stability of themembrane to chemical and water permeability depends on the integrity ofthe lipid bilayer and on tight control of temperature, pH, andosmolarity which are disrupted by ischemia. Lowering the temperaturecauses a phase transition of lipids and results in profound changes inmembrane stability. In addition, it drastically alters the function ofmembrane-bound enzymes. Hypothermia-induced structural changes in themembrane increase permeability, which contributes to cell swelling.Therefore, organ-preservation solutions are hypertonic to minimize thesealterations.

Until relatively recently, the primary solution used for cold-storagepreservation of the kidneys was Euro-Collins solution. Its formulationprovides a hyperosmolar environment with an intracellular electrolytecomposition intended to reduce cellular swelling. In combination withhypothermia, kidneys can be safely stored in this solution for up to36-48 hours before transplantation. Groenewoud A F, Thorogood J. Apreliminary report of the HTK randomized multicenter study comparingkidney graft preservation with HTK and EuroCollins solutions. HTK StudyGroup. Transpl Int. 1992; 5 Suppl 1:S429-32.

There are currently two modes of preservation methods for kidneys andlivers: static and dynamic. Simple cold storage (SCS) is the main methodfor static storage while hypothermic machine perfusion (HMP),normothermic machine perfusion (NMP) and oxygen persufflation (OP)comprise the methods for dynamic preservation. Of these four methods,only SCS and HMP are approved clinically for kidneys and only SCS forlivers. The remaining methods are in various stages of pre-clinical andearly clinical studies. The first successful human preservation of akidney for 17 hours: Lee, et al, Organogenesis. 2009 July-September;5(3): 105-112. Hypothermic machine perfusion (HMP) preservation.Hypothermic machine perfusion was developed for kidneys to extend bothpreservation time and preservation quality.

The kidney is the most widely transplanted organ with the longesthistory of preservation research. Simple SCS can reliably provide goodearly function in the majority of grafts where storage times over 36 hhave not been required within modern integrated transplant networks.Revolutionary new designs for kidney perfusion machines and a shifttowards marginal donors led to a re-evaluation of HMP. A prospectivemulti-centre trial has demonstrated a clear outcome benefit for bothdelayed graft function and graft survival at 1 year. The perfusate usedwas a formulation of University of Wisconsin (UW) solution, wherelactobionate was replaced by gluconate. The introduction of UW solutionallowed good preservation of liver to be extended to around 15 h.Guibert, E., et al., Transfus Med Hemother. April 2011; 38(2): 125-142.

The mainstay of clinical lung preservation has been simple hypothermicimmersion of inflated lungs following flush of the pulmonary artery withchilled preservation solution. Low potassium solutions such as theWallwork solution have been favoured. The routine application of lunginflation during preservation renders the lungs capable of some aerobicmetabolic activity and has led to increased interest in including NHBDdonor lungs for clinical preservation. There are occasions where lungsand heart are cold preserved en bloc using cold carioplogic arrest ofthe heart followed by pulmonary flush of a second preservation solution(such as Euro-Collins solution) for the lungs. Guibert. E. et al.(2011).

There are two steps in cardiac SCS. First cardiac activity is stopped ina controlled fashion by flushing the vascular bed of the organ withchilled cardiaplegic solution to cool and wash out the blood. Solutionssuch as St. Thomas' cardiaplegic solution are used in the majority ofcentres. Then hearts are generally also topically cooled with chilledsterile saline before packing and storage in ice to provide satisfactorypreservation in the range of 4-6 h. Instead of storing and packing insaline, the n-3 glyceride perfusion emulsions of the present inventioncan be used to improve preservation.

In principle, cold flush storage or preservation is based upon thepremise that temperature reduction to near but not below the ice point(e.g., about 0° C.) precludes the need to support metabolism to anysignificant extent, and that the correct distribution of water and ionsbetween the intracellular and extracellular compartments can bemaintained by physical rather than metabolic means. During a period thatmetabolic pumps are inactivated, the driving force for transmembrane ionflux is the difference in ionic balance between intracellular andextracellular fluid. The driving force for water uptake (cell swelling)is the impermeant intracellular anions. Thus changes can be prevented orrestricted by manipulating the extracellular environment to abolishchemical potential gradients. On this basis, a variety of flush, ororgan and tissue washout solutions have been devised and evaluated forcold storage. These solutions are often referred to as “intracellular”solutions due to their resemblance, in some respects, to intracellularfluid. The n-3 perfusion/reperfusion emulsions are types of washout,preservation, intracellular and flush solutions.

Generally, the two most frequently used methods for preserving organsafter removal from the donor are simple hypothermic storage andcontinuous pulsatile perfusion. With simple hypothermic storage, theorgan is removed from the donor and cooled rapidly. This is usuallyachieved by a combination of cooling and short periods of perfusion todrop the organ temperature as quickly as possible to a temperaturebetween 0° C. and 4° C. where it may be held for up to about six hoursor significantly longer, even up to 72 hours in some cases. The durationdepends in part on the organ or tissue. While cold storage enablesorgans to be transplanted, the time during which the organ is viable isshort. Cold storage decreases the rate at which intracellular enzymes,essential cellular components necessary for organ viability, degrade butdoes not stop metabolism entirely.

The second method of organ preservation which has undergone extensiveinvestigation, continuous pulsatile perfusion, utilizes the followingelements: (1) pulsatile flow, (2) hypothermia, (3) membrane oxygenation,and (4) a perfusion buffer. Although being more technically complex andcostly, continuous pulsatile perfusion provides significantly longerviability of the organ when compared to simple hypothermia.

Preserving organs at between 0° C. and 4° C. can result in damage to theorgan during storage and upon reperfusion, with a warm reperfusionsolution. Injury to the organ occurs through damage to epithelial andendothelial cells. Although some of the solutions of the prior art havebeen useful to extend the storage time of donor organs and lessen injuryto the organ upon reperfusion, cell injury still does occur frequently.It is desirable to extend the viable organ life and improve the qualityof the transplanted organ. Extending organ viability allows sufficienttime for compatibility testing of the donor and recipient, and increasedorgan availability. Ischemia-reperfusion injury to transplanted organspreserved in solutions of the prior art is still often associated withloss of viability such as delayed graft function, and predisposition toacute and chronic rejection.

The principle design elements of organ and tissue perfusion/reperfusionbuffers include adjusting the ionic balance (notably of the monovalentcations) and raising the osmolality by including an impermeant solute tobalance the intracellular osmotic pressure responsible for water uptake.

Prior to 1988, a standard solution for clinical preservation of organsand tissues was Collins solution, which includes potassium phosphate,magnesium sulfate and glucose. In recent years, however, this has beensuperseded either by a modified version called “Euro-Collins” in whichthe magnesium sulfate is omitted, or more extensively by the Universityof Wisconsin solution (UW solution) in which much of the phosphate anionhas been replaced with lactobionate, and in which glucose has beenreplaced with raffinose. These larger molecules appear to improveprotection against adverse effects of cell swelling during hypothermicstorage, as compared to prior solutions.

Preservation solutions are designed to prevent or inhibit variousmechanisms which cause injury to the organ, and thus they perform one ormore (and preferably all) of the following functions: (1) prevent orrestrict intracellular acidosis, (2) prevent the expansion ofintracellular space, (3) prevent injury from oxygen-derived freeradicals, especially during reperfusion, (4) enable the regeneration ofhigh-energy phosphate compounds during reperfusion, (5) sustainappropriate metabolic requirements and prevent the rapid changes inintracellular ions including N⁺, —H⁺ and Ca²⁺ following reperfusion.

4. SUMMARY OF RESULTS AND EMBODIMENTS OF THE INVENTION

The following is a summary of results of experiments described in theExamples of this application.

Perfusion of Isolated Hearts Ex Vivo with n-3 Glyceride PerfusionEmulsions Reduced Reperfusion Damage after an Ischemic Event (Details inExample 2)

-   -   An ex vivo model using hearts removed from C57BL/6 mice and        perfused in the well-known Langendorff system was used. After a        period of 30 min of induced ischemia, hearts were reperfused for        1 hour. Control hearts reperfused perfusion buffer without n-3        triglycerides, showed that heart rate was reduced, LVDP was        markedly decreased, and arrhythmias were observed. Test hearts        were reperfused in perfusion buffer with n-3 triglycerides (“n-3        TG perfusion emulsion”) which maintained a normal heart rate,        normal LVDP, and dramatically reduced the occurrence of        arrhythmias. Hearts perfused with n-3 triglyderides had close to        100% (or normal) recovery.    -   After 1 hour of reperfusion with the test n-3 triglyceride        reperfusion buffer, there was over a 90% recovery of LVDP        compared to only a 40% LVDP recovery in the control reperfusion        buffer. In addition, heart rate recovery was improved with n-3        triglyceride reperfusion buffer;    -   Markers of myocardial infarction such as creatine kinase were        markedly decreased in the reperfusion of isolated hearts in the        test n-3 triglyceride reperfusion buffer. Increases in level of        the anti-apoptotic gene marker, Bcl-2, and in expression of        Bcl-2 mRNA were also shown. In contrast, both the level of        protein and mRNA for HIF1, a marker for autophagy which may        increase cell death, were down-regulated after reperfusion with        the test n-3 triglyceride reperfusion buffer.    -   Another autophagic marker, Beclin 1, was also decreased in terms        of protein expression and mRNA expression after reperfusion with        buffer containing n-3 glycerides.    -   PPAR gamma protein expression, associated with pro-inflammatory        responses in heart, is markedly increased after reperfusion in        control hearts but decreased after reperfusion containing n-3        glycerides.    -   To summarize, including n-3 glycerides in the perfusion buffer        during reperfusion after induced ischemia decreased the marker        of myocardial infarction creatine kinase, and upregulated        beneficial proteins including the anti-apoptotic gene marker        Bcl-2. Reperfusion with perfusion buffer including N-3 TG        downregulated of harmful proteins including HIF1 (a marker for        autophagy which may increase cell death), PPAR gamma (associated        with pro-inflammatory responses in heart), and Beclin 1 (an        autophagic marker) were down-regulated.        Administration of n-3 TG Emulsions In Vivo Reduced Reperfusion        Injury after H/I (Details are in Examples 3 and 4)    -   As used herein “Tri-DHA” means n-3 triglyceride emulsions rich        in DHA having at least 30% DHA; often over 90% of the TG fatty        acids are DHA. TG90-DHA30 emulsion that was administered in vivo        following H/I to reduce reperfusion injury at a concentration of        10% omega-3 fish oil by weight in grams per 100 ml of emulsion,        wherein the omega-3 oil is >90% triglyceride (TG) by weight per        total weight of the omega-3 oil, and in which up to about 30% of        the total acyl groups of the TG are DHA.    -   Effects of in vivo administration of n-3 TG emulsions rich in        DHA (specifically “TG90-DHA30”) on blood triglyceride levels        after injection indicated that TG levels were substantially        increased up to three-fold higher at 1.5 hours compared to        baseline, followed by a decrease of levels to baseline at 3 and        5 hours due to the fact that n-3 TG had entered into the blood        stream and was being catabolized.    -   After H/I it was determined that there was no difference in        blood glucose levels among TG90-DHA30 vs. n-6 TG vs. saline        control; and no difference was observed in capillary bleeding        times in n-3 TG90-DHA30 treated mice as compared to saline        controls. FIG. 17.    -   n-3 TG did not change cerebral blood flow after H/I since very        similar blood flow levels were maintained in neonatal H/I mice        whether they were saline-treated or n-3 TG90-DHA30-treated.    -   a n-3 TG90-DHA30 but not n-6 TG protected the brain against H/I        injury as evidenced by the subcortical region of the brain where        infarct volume was substantially decreased in n-3 TG treated        mice but a significant increase in infarct volume occurred with        n-6 TG emulsion injection.    -   In immediate post-H/I treatment the total brain infarct area was        significantly reduced almost 50% in the n-3 TG90-DHA30 post H/I        treated group.    -   DHA but not EPA was neuroprotective after H/I as evidenced by        total infarct size reduction by a mean of 48% and 55% with        treatment of 0.1 and 0.375 g TG wherein the DHA=99% of the total        acyl groups on the TG, respectively, compared with saline        control. However, neuroprotection was not observed with Tri-EPA        injection wherein the EPA=99% of the total acyl groups on the TG        at either of the two doses compared with saline treatment.    -   No protective effect from TG90-DHA30 after a 4-hour delay in        treatment was observed compared with control, but TG90-DHA30        administered at 0 hour immediately post-H/I, and then delayed        1-hr, and 2-hr post stroke showed similar reduced brain infarct        volumes (˜50%) compared to control.    -   Effects of H/I and TG90-DHA30 treatment on brain and neuronal        cell loss were measured for long-term outcome at 8 weeks after        H/I insult and it was found that neuroprotection after injury        and TG90-DHA30 injection that was observed 24 hours after H/I        can be seen histologically approximately 2 months after the        initial stroke insult.

5. METHODS OF PERFUSION AND REPERFUSION

The n-3 glyceride perfusion emulsions of the present invention aredesigned to preserve and store the organ (or tissue) with thepreservation solution and reperfuse with preservation solution prior toimplantation. Typically, the surgeon removes the organ and connects itto a perfusion apparatus comprising tubing and pumps. The preservationsolution is then perfused through the organ while gassed with oxygen andcarbon dioxide while it is awaiting implantation into a patient. Aperfusion rate of from about 25 to about 150 mL/hour, or about 50mL/hour, at 1° C. has been found to be effective. However, routineexperimentation can optimize this process and varies according to thetemperature of duration of organ/tissue storage. Organ perfusion canoccur at either a constant flow or pressure.

The preservation solution can be used at all temperatures ranging from0° C. to normal body temperature, 37° C. The duration ofperfusion/reperfusion and the temperature is selected in order tooptimize the process of sustaining, maintaining or improving theviability of the organ/tissue while being stored, for example beforeand/or during transplantation. In an embodiment of a method of thepresent invention, organs/tissues can be used at predominatelyhypothermic temperatures, to provide a decrease in organ metabolism,lower the energy requirements, delay the depletion of high energyphosphate reserves and accumulation of lactic acid and retard themorphological and functional deterioration associated with disruption ofblood supply. Alternatively, a method for storing a particular organ maybe selected that first employs mid-normothermic to normothermictemperatures to improve the viability of the organ and/or tissue untilan organ and/or tissue meeting a predetermined threshold viability indexis obtained (which may vary from organ to organ), thereafter hypothermictemperatures may be employed to provide a decrease in organ metabolism,lower the energy requirements, delay the depletion of high energyphosphate reserves and accumulation of lactic acid and retard themorphological and functional deterioration associated with disruption ofblood supply.

Exemplary organ perfusion methods are described in US 20040237693; US20090311663; US 20110173023; 20120315618; and 20100278934.

Organ perfusion apparatus are available to perfuse one or more organssimultaneously, at normothermic, near-normothermic, mid-normothermic andhypothermic temperatures (hereinafter, normothermic, near-normothermic,mid-normothermic and hypothermic perfusion modes).

Plasma-like electrolytes as base for oxygen carrying molecules and othersubstrates are sometimes necessary for optimized “normothermic”perfusion

Cryo-Concentrated Intracellular Base plus permeating or non-permeatingcryoprotective additives can be used for sub-zero preservation of cellsand tissues.

In embodiments, multiple n-3 glyceride perfusion/reperfusion emulsionsthat are not identical can be perfused/reperfused into the organ ortissue.

In embodiments, the n-3 glyceride perfusion emulsions areadministered/perfused to the organ (or tissue) while it is maintained ina predetermined temperature range, such as from about 10° C. to about37° C., or from about 15° C. to about 37° C., or from about 20° C. toabout 37° C., or from about 20° C. to about 30° C., or from about 20° C.to about 25° C.

Ex vivo perfusion of an isolated organ (or tissue) includes treatment ofthe organ (or tissue) that has endured a period or periods of ischemiaand/or apoxia with the n-3 glyceride emulsions of the invention. Ex vivotreatments may involve performing surgical techniques on an organ, suchas cutting and suturing an organ, for example to remove necrotic tissue.

The above methods may be used for mammalian organs, including humans,including small organs from a child as well as large or adult organs.

The optimum perfusate regimen/therapy and/or temperature mode formaintaining, restoring, and improving the viability of isolatedorgans/tissues in order to meet a predetermined threshold viabilityindex, may vary from organ to organ and tissue to tissue, and may dependon the condition or state the organ/tissue was received in (e.g.,differences include heart-beating donors versus non-heart beating donorsand amount of time the organ has been out of the body).

In embodiments, depending on the particular organ therapy decision andthe decision regarding whether to transplant the organ, normothermicperfusion may be preceded by and/or followed by hypothermic perfusion,near-normothermic perfusion, or mid-normothermic perfusion orcombinations thereof. Such perfusion conditions may also be employed invivo as well as in vitro prior to removal of the organ from the donor.

6. OMEGA-3 OILS

Lipid generally refers to a group of natural substances which aresoluble in hydrocarbon and insoluble in water. Lipids include anyfat-soluble (hydrophobic) naturally-occurring molecules. The term ismore specifically used to refer to fatty-acids and their derivatives(including tri-, di-, and monoglycerides and phospholipids) as well asother fat-soluble sterol-containing metabolites such as cholesterol.

Chemically, fatty acids can be described as long-chain monocarboxylicacids the saturated examples of which have a general structure ofCH3(CH2)nCOOH. The length of the carbon chain usually ranges from 12 to24, always with an even number of carbon atoms. When the carbon chaincontains no double bonds, it is a saturated chain. If it contains one ormore such bonds, it is unsaturated. The presence of double bonds reducesthe melting point of fatty acids. Furthermore, unsaturated fatty acidscan occur either in cis or trans geometric isomers. In a vast majorityof naturally occurring fatty acids, the double bonds are in thecis-configuration.

Polyunsaturated fatty acids (PUFA) include omega-6 (also known as ω-6 orn-6) and omega-3 (also known as ω-3 or n-3) polyunsaturated fatty acids.The designation as omega-3 or omega-6 is based on the fatty acidstructure, namely the distance of the first unsaturated bond from themethyl (omega) end of the fatty acid molecule. Omega-3 polyunsaturatedfatty acids mainly include cis-20:5(Δ5,8,11,14,17)-eicosapentaenoic acid(EPA); cis-22:5(Δ7,10,13,16,19)-docosapentaenoic acid (DPA);cis-22:6(Δ4,7,10,13,16,19)-docosahexaenoic acid (DHA); andcis-18:3(Δ3,6,9)-α-linoleic acid. Omega-6 polyunsaturated fatty acidsmainly include cis-18:2(Δ9,12)-linoleic acid and cis-20:4(Δ5, 8, 11,14)-arachidonic acid.

Glycerol is a chemical compound with the formula HOCH2CH(OH)CH2OH.Glycerides are lipids possessing a glycerol (a crude name for which ispropan-1, 2, 3-triol) core structure with one or more fatty acyl groups,which are fatty acid-derived chains attached to the glycerol backbone byester linkages.

A diglyceride (“DG”), also known as a diacylglycerol, is a glycerideconsisting of two fatty acid chains covalently bonded to a glycerolmolecule tough ester linkages. A triglyceride (“TG”) (also known astriacylglycerol or triacylglyceride) is a glyceride in which theglycerol is esterified with three fatty acids. An acyl group is afunction group derived by the removal of one or more hydroxyl group andoxoacid.

Triglycerides (“TG”) may also be classified as having a long or mediumchain length. Long chain triglycerides preferably contain fatty acidswith 14 or more carbons, while medium chain triglycerides preferablycontain fatty acids with 6 to 12 carbons. Long chain triglycerides mayinclude omega- and omega-6 fatty acids. Medium chain triglycerides havesaturated fatty acids and thus do not contain omega-6 or omega-3 fattyacids. Long chain triglycerides (LCT) and medium chain triglycerides(MCT) may serve as energy sources. Medium chain triglycerides mayinfluence the metabolism of emulsion droplets because of their fasthydrolysis and other properties (i.e. enhancing particle binding tocells, changing cell membrane properties).

The human body is capable of synthesizing certain types of fatty acids.However, long chain omega-3 and omega-6 are designated as “essential”fatty acids because they cannot be produced by the human body and mustbe obtained through other sources. For example, fish oils fromcold-water fish have high omega-3 polyunsaturated fatty acids contentwith lower omega-6 fatty acid content. Table 1 was supplied by themanufacturer of the n-3 Tri-DHA oil Fresenius Kabi, and it describes themake-up of the n-3 Tri-DHA used in some of the experiments describedherein. Table 1 estimates (in column 2) that the n-3 fish oil comprisesa range of 1-7% in gm/100 ml Linoleic acid (C18:2n-6), and 1-4%Arachidonic acid (C20: 4n-6), which together have a theoretical upperlimit of 11%. However, to date no fish oil used as a major source ofomega-3 fatty acids has been reported that has over 10% omega-6 fattyacids. The di- and tri-glyceride omega-3 emulsions of the presentinvention when made from fish oil, use fish oil with 10% or less omega-6fatty acid. Most vegetable oils (i.e., soybean and safflower) have highomega-6 polyunsaturated fatty acids (most in the form of 18:2(Δ^(9, 12))-linoleic acid) content but low omega-3 (predominantly18:3(Δ^(9, 12, 15))-α-linolenic acid) content.

Methods of synthesizing di- and triglycerides rich in n-3 fatty acidsare well known in the art, and are disclosed, for example, in U.S. Pat.Nos. 2,206,168; 2,626,952; 3A10, 881; 3,634,473; 3,097,098; 3,551,464;4,018,806; 5,106,542; 5,130,061; 5,142,071; 5,142,072; 5,959,128;5,434,280; 6,004,611; 6,337,414; 6,537,787; 6,749,881; and 7,081,542 allof which are incorporated herein by reference. Thus, the diglyceridesand triglycerides may be obtained by trans-esterification of variousoils (such as fish oil or rapeseed oil) containing omega-3 unsaturatedacyl-groups, and/or monoenoic acyl-groups with glycerol. Di- andtriglycerides may also be obtained by esterification of a fatty acidderived from such an oil with glycerol. In the n-3 glyceride perfusionemulsions of the present invention, the omega-3 di- and triglyceridesare typically derived from fish oil, or synthesized so as to containless than about 3% omega-6 fatty acids. Fish oils include natural fishoils, processed fish oils, highly purified fish oil concentrates or(re)esterified (synthetic) fish oils, including (re-)esterification ofomega-3-fatty acids from cold water fish oil by triglyceride hydrolysis,purification and concentration of the resultant omega-3-fatty acids withglycerol. Processed fish oils are described in European published patentapplication EP-A-0298293, which is incorporated herein by reference inits entirety.

Suitable exemplary fish oils include oils from cold-water fish such assalmon, sardine, mackerel, herring, anchovy, smelt and swordfish. Fishoils generally contain glycerides of fatty acids with chain lengths of12 to 22 carbons. Highly purified fish oil concentrates obtained, forexample, from sardine, salmon, herring and/or mackerel oils may have aneicosapentaenoic acid (EPA) content of from about 9-10 or up to ˜20% oftotal TG fatty acids, and a docosahexaenoic acid (DHA) content of 10% upto ˜20% based on the fatty acid methyl esters analyses of the fish oilconcentrate as determined by gas chromatography (percent by area). U.S.Pat. No. 6,159,523, incorporated herein by reference in its entirety,discloses a method for making fish oil concentrates. Generally, theamount of the polyunsaturated fatty acids of the omega-6 series (such aslinoleic acid) in natural fish oils is low, i.e. less than 10%,typically less than 5% of total fatty acids by weight.

Also, trans-esterification reactions may be performed by chemical means(such as using an alkali catalyst, i.e. sodium methoxide). Or, di- andtriglycerides may be prepared by enzymatic approaches with lipases. Theresulting glyceride may be further processed by isomerase to yield a 1,2or 1,3-glyceride.

Omega-3 EPA and DHA may be obtained from any source. For example, EPA orDHA may be synthetic, isolated from natural products such as krill oil,or obtained from fish oil by alkaline hydrolysis. Fish oil is currentlyand generally the least expensive source of EPA and DHA.

Based on the total amount of acyl groups, in certain embodiments atleast about 20% to 99% of the total acyl-groups of the omega-3diglycerides or triglycerides comprise EPA or DHA.

In other embodiments, the new omega-3 glyceride perfusion emulsions ofthe present invention may further comprise from about 0% to about 10% ofmonoglycerides of DHA and/or EPA. The monoglycerides of DHA and/or EPAare from about 0% to about 10%, or from about 0% to about 2%, based onthe total amount of lipid.

New omega-3 glyceride perfusion emulsions of the present invention mayalso further comprise from about 0% to about 20% total free unsaturatedfatty acids by weight. Typically the unsaturated fatty acids are fromabout 0% to about 5%, or from about 0% to about 2%, based on the totalamount of fatty acids by weight in the lipid phase. In certainembodiments of the invention, omega-3 glyceride perfusion emulsionscomprise medium chain triglycerides (MCT). These omega-3 glycerideperfusion emulsions may contain as a percent of total n-3 lipid, andfrom 0% to 90% medium chain di- or triglycerides of total glycerides, orfrom about 0% to about 60%, or from about 40% to about 60% of totalglycerides. Medium chain di- or triglycerides may contain fatty acidswith 6 to 12 carbons. The medium-chain triglycerides (“MCT”)administered with the lipid emulsions serve mainly as a source ofenergy; they contain saturated fatty acids and hence contain neither theomega-6 nor omega-3 essential fatty acids. Because of their fasthydrolysis as well as other properties (enhancing particle binding tocells), MCT may have a positive effect on the metabolism of emulsionparticles.

7. PHARMACEUTICAL FORMULATIONS OF N-3 GLYCERIDE PERFUSION EMULSIONS

Omega-3 glyceride perfusion emulsions of the present invention have lessthan 7% of an omega-3 oil by weight in grams per 100 ml of perfusionbuffer, with embodiments ranging from about 0.5 to about 1%, 1-3%, 3-5%or 5-less than 7% omega-3 oil weight in grams per 100 ml of perfusionbuffer. In the ex vivo heart example, herein, the amount was only 0.3%.Significantly higher % n-3 emulsions (7-35% n-3 oil weight in grams per100 ml of perfusion buffe) are used for in vivo administration to reducereperfusion injury after H/I because the concentration is diluted in thebody. Organs and tissues stored in the n-3 glyceride perfusion emulsionsof the present invention can be perfused/reperfused with emulsionshaving much lower n-3 oil content.

The omega-3 oil in the new perfusion emulsions comprises from about 10%to about 99% omega-3 diglyceride, omega-3 triglyceride or combinationsthereof by weight per total weight of the omega-3 oil. However, thisamount can vary widely. For Example the Tri-DHA emulsions administeredin vivo in Example 3 had >90% of TG fatty acids as DHA. In certainembodiments the amount of TG or DG ranges from about 10% to about 20%,from 20% to about 40%, from 40% to about 60% and from 60-99%. About 20%to about 99% of the total acyl groups of the omega-3 of the diglyceridesor triglycerides consist of EPA or DHA, and this can range from about20% to 50%, from 50% to 75%, from 75% to 90%, from 90% to 97% of thetotal acyl groups of the omega-3 diglycerides or triglyceridesconsisting of EPA or DHA.

Omega-3 glyceride perfusion emulsions of the present invention maycontain a stabilizing or isotonizing additive; usually glycerol isadded. Preferred stabilizing or isotonizing additives include glycerol,sorbitol, xylitol or glucose. Glycerol is most preferred.

In addition to perfusion buffer, omega-3 glyceride perfusion emulsionsmay contain conventional auxiliary agents and/or additives, such asemulsifiers, emulsifying aids (co-emulsifiers), stabilizers,antioxidants, and isotonizing additives. Emulsifiers may includephysiologically acceptable emulsifiers (surfactants) such asphospholipids of animal or vegetable origin. Examples of phospholipidsare egg yolk lecithin, a biologic phospholipid, a phosphatidylcholinewith fixed fatty acyl chain composition, a glycophospholipid or aphosphatidylethanolamine. Other lecithins, such as soy lecithin may beused. Particularly preferred are purified lecithins, especially soybeanlecithin, egg lecithin, or fractions thereof, or the correspondingphosphatides. The emulsifier content may vary according to industrystandards from about 0.02% to about 2.5%, or from about 0.6% to about1.5% and most of about 1.2%, based on the total weight of the emulsion.In one embodiment the emulsifier is 1.2 mg of egg yolk lecithin/I 00 mlemulsion.

Alkali metal salts, such as sodium salts, of long chain, C₁₆ to C₂₈fatty acids may also be used as emulsifying aids (co-emulsifiers). Theco-emulsifiers are employed in concentrations of from about 0.005% toabout 0.1%, or about 0.02% to about 0.04%, based on the total weight ofemulsion. Further, cholesterol may be added in combination with otherco-emulsifiers may be employed as an emulsifying aid in a concentrationof from about 0.005% to about 0.1%, or from about 0.02% to about 0.04%,based on the total weight of emulsion.

Omega-3 glyceride perfusion emulsions may further comprise an effectiveamount of an antioxidant, such as vitamin E, in particular a-tocopherol(the most active isomer of vitamin E in humans) as well as gammatocopherol, and/or ascorbyl palmitate as antioxidants and thus forprotection from peroxide formation. The total amount of alpha tocopherolmay be up to 1000 mg per liter. In a preferred embodiment the totalamount of said antioxidant is from about 10 mg to about 1000 mg, more orfrom about 25 mg to about 1000 mg, or from about 100 mg to 500 mg, basedon 100 g of lipid.

Preparation of the n-3 glyceride perfusion emulsions are known in theart. Omega-3 lipid-based perfusion emulsions according to the inventionare oil-in-water (o/w) emulsions in which the outer continuous phase isthe perfusion buffer purified or sterilized for use to storeorgans/tissues. Such n-3 glyceride perfusion emulsions may be obtainedby standard methods, i.e. by mixing the oil components followed byemulsification and sterilization. The pH value of the lipid emulsion maybe adjusted to a physiologically acceptable value, preferably to a pH offrom about 6.0 to about 9.0, more preferably from about 6.5 to about8.5. Auxiliary agents and additives may be added to the oil mixtureprior to emulsification or prior to sterilization.

Omega-3 glyceride perfusion emulsions according to the invention can beprepared by known standard procedures. Typically, first the lipids,emulsifier and other auxiliary agents and additives are mixed and thenfilled up with water with dispersing. The water may optionally containadditional water-soluble components (e.g. glycerol).

Lipid particles in the perfusion emulsions of the present invention mayhave a median particle size of less than 1 μm, more preferably 100 to500 nm.

Examples below are perfusion buffers available currently that can beused in the n-3 glyceride perfusion emulsions of the present inventionand related methods. Marketed Products:

AQIX® RS-I Solution™ (Aqix Ltd) AQIX® RS-I solution uniquely replicatesthe tissue fluid present in all human organs, without adding extracts ofhuman or animal serum for donor organ transplantation perfusion. AQIX®RS-I is in pre-clinical development ahead of CE marking as a Class 3medical device for use in the transport, storage and/or functionalevaluation of organs prior to transplantation.

Machine Perfusion Solution-Belzer UW™ (Bridge to Life Ltd) MachinePerfusion Solution-Belzer UW® is intended for the in vitro flushing andtemporary continuous machine perfusion preservation of explantedkidneys.

STEEN Solution™ (XVIVO Perfusion AB) The circuit perfusion of the lungmimics in-vivo conditions; the ventilated lung is perfused with a 15%deoxygenated suspension of red cells in STEEN Solution™ and the criticalparameters of gaseous exchange, pulmonary vascular resistance and otherkey variables under normothermic conditions are monitored.

Liver Perfusion Medium™ (Life Technologies Corporation) Liver PerfusionMedium is a buffered, balanced salt solution formulated to cleanse theliver of blood, prevent clotting, and initiate loosening of cell-to-cellcontact.

Other perfusion/reperfusion solutions are described in: 20090311663;U.S. Pat. Nos. 4,879,283 and 4,798,824. These patents cover the widelyused organ preservation solution commercially available under the tradename VIASPAN™ marketed by Barr Laboratories.

Additionally, other agents that assist in conserving and preparing anorgan or tissue for transplant may be added to an n-3 glycerideperfusion/reperfusion emulsion of the present invention such as any oneor more of the following agents: antibiotics, VEGF, KGF, FGF, PDGF, TGF,IGF-1, IGF-2, IL-1, prothymosin and/or thymosin 1 in an effectiveamount. Other buffers include:

Phosphate-Buffered Sucrose Solution

This solution contains sucrose 140 mmol/L and sodium hydrogen anddihydrogen phosphate as buffers. In experimental studies, it preserveddog kidneys for 3 days. It is not commonly used today.

University of Wisconsin Solution

University of Wisconsin (UW) solution was developed for liver, kidney,and pancreas preservation. It has been considered the standard for renaland hepatic preservation, effectively extending the ischemic time forkidneys and livers and allowing them to be transported considerabledistances to waiting recipients. UW solution has also been successfullyapplied to small-bowel and heart preservation. The composition of thesolution is complex. Analysis of its various components has shown thatsome may be omitted or replaced with results similar to that of theoriginal solution. The solution has an osmolality of 320 mmol/kg and pH7.4 at room temperature and is composed of the following:

-   -   Potassium 135 mmol/L    -   Sodium 35 mmol/L    -   Magnesium 5 mmol/L    -   Lactobionate 100 mmol/L    -   Phosphate 25 mmol/L    -   Sulphate 5 mmol/L    -   Raffinose 30 mmol/L    -   Adenosine 5 mmol/L    -   Allopurinol 1 mmol/L    -   Glutathione 3 mmol/L    -   Insulin 100 U/L    -   Dexamethasone 8 mg/L    -   Hydroxyethyl starch (HES) 50 g/L

Bactrim 0.5 ml/LHES conveys no advantage to the solution when used forcold storage, and it, in fact, adds to the viscosity of the solution aswell as to the expense. HES-free derivatives of the solution have givensimilar, if not better, clinical results than those of the originalformulation. The lactobionate is the major effective component of thesolution. Its insoluble nature maintains the colloid oncotic pressure ofthe solution, delaying or preventing equilibration of the solutionacross the cell membrane, and thus delaying the development of cellularedema. The lowered concentration of potassium improves the flushingefficiency of the solution by removing the vasoconstrictive effect ofthe high potassium solution. Glutathione is unstable in solution andeffective as an oxygen free-radical scavenger only if it is addedimmediately before use. Adenosine and allopurinol help in this function.

Celsior Solution

Celsior is a recently developed extracellular-type, low-viscosity (dueto the absence of HES) preservation solution that couples theimpermeant, inert osmotic carrier from UW solution (by usinglactobionate and mannitol) and the strong buffer from Bretschneider HTKsolution (by using histidine). The reduced glutathione in Celsiorsolution is the best antioxidant available. The solution wasspecifically designed for heart transplantation. It is being currentlyused in clinical lung, liver, and kidney transplantations and it isunder investigation for pancreas transplantation.

The contents of Celsior solution are as follows:

-   -   Sodium 100 mmol/L    -   Potassium 15 mmol/L    -   Magnesium 13 mmol/L    -   Calcium 0.25 mmol/L    -   Lactobionate 80 mmol/L    -   Glutathione 3 mmol/L    -   Glutamate 20 mmol/L    -   Mannitol 60 mmol/L    -   Histidine 30 mmol/L        Kyoto ET Solution

Researchers at Kyoto University developed a new solution that contains ahigh sodium concentration, a low potassium concentration, trehalose, andgluconate. The solution is chemically stable at room temperature. It isbeing investigated for skin storage and lung preservation in a ratmodel. ET Kyoto solution is also being actively investigated in clinicaltrials for transplantation of the lungs, heart, and other organs.

Its constituents include the following:

-   -   Sodium 100 mmol/L    -   Potassium 44 mmol/L    -   Phosphate 25 mmol/L    -   Trehalose 41 mmol/L    -   HES 30 gm/L    -   Gluconate 100 mmol/L

8. EXAMPLES Example 1: Materials and Methods

All research studies were carried out according to protocols approved bythe Columbia University Institutional Animal Care and Use Committee(IACUC) and in accordance with the Association for Assessment andAccreditation of Laboratory Animal Care guidelines.

General Method of Preparation of a Diglyceride (DG) and Triglyceride(TG) Emulsions.

Phospholipid-stabilized emulsions of n-3 (DG and/or TG) are typicallyprepared with fish oil DG and/or TG (synthetic oils can also be used),and egg yolk phospholipid. In an embodiment the amount of n-3 oil in theperfusion emulsions is as low as 0.05%.

Each emulsion contained the desired amount of n-3 oil, a diglyceride ora triglyceride, which is typically emulsified by 1.2 g of egg yolklecithin, and 2.5 g of glycerol/100 mL water. The emulsion lipids aremixed in doubly distilled water (30 g of water) and dispersed by meansof an Ultra-Turrax (Janke and Kunkel K G, Staufen, West Germany) for 10min; water is added to give a final volume of 100 mL, and emulsions aredispersed for an additional 10 min. Subsequently, the dispersion ishomogenized by ultrasound in a cooling cell with a Labsonic 2000homogenizer for 10 min at an energy input of 200 W. The emulsions arethen sealed in 5 mL vials under N2, and thereafter kept at 4° C. in someprocesses for industrial production emulsion volumes of up to 3-500 mlmay be made. Mean particle sizes are determined by laser spectroscopy,and are similar in both size and homogeneity with mean diameters between290 and 300 nm. If the emulsions obtained still contain lipid particleshaving a diameter that is too large, the average droplet sizes ofemulsions may be further reduced by additional homogenization, e.g., byusing a high-pressure homogenizer.

Animal Care

All studies in heart were performed with the approval of theInstitutional Animal Care and Use Committee at Columbia University, andNew York University School of Medicine, and conform to the Guide for theCare and Use of Laboratory Animals published by the US NationalInstitutes of Health (NIH Pub. No. 85-23, 1996). C57BL6 mice (weightbetween 25-30 g and 12-14 weeks old) were obtained from JacksonLaboratories for our studies. Mice were kept in an animal care facilityfor a week prior to the studies. All mice were fed a normal chow diet(Teklad Global diets, Harlan Laboratories).

Reagents

The primary antibodies used were Bcl-2, Beclin-1, PPAR-γ, p-AKT,total-AKT, p-GSK-3β, total-GSK-3β (Cell Signaling, USA); and β-actin (BDBiosciences Pharmingen, USA). The secondary antibodies used wereanti-rabbit IRdye800, anti-mouse IRdye700 (1:50,000 dilution). SB216763(3 μM), Rosiglitazone (6 mg/kg body weight) were purchased fromSigma-Aldrich, USA. Phosphatidylinositol 3-kinase (PI3K)/AKT inhibitorLY-294002 (10 μM) was purchased from Calbiochem. The doses of theinhibitors and agonist used in this study were based on publications inthe literature.¹ n-3 fish oil-based emulsion (10 g of TG/100 mL) wascommercially prepared intravenous phospholipid-stabilized emulsions, andcontained high concentrations of n-3 FA as previously described^(2,3)n-3 TG emulsion was rich in EPA (up to 28%) and DHA (up to 30%).

Example 2: Ex Vivo Ischemia and Reperfusion with n-3 Glyceride PerfusionEmulsion

A. Materials and Methods

Experiments were carried out and modified for use in mice hearts.^(1,2)C57BL6 mice weighting between 25-30 g and 12-14 weeks old wereanesthetized by injecting ketamine/xylazine cocktail [80 mg/kg and 10mg/kg respectively]. The hearts were rapidly excised and then wereretrograde perfused through the aorta in a non-recirculating mode, usingan isovolumic perfusion system through Langendorff technique (LT), withKrebs-Henseleit buffer, containing (in mM) the following: 118 NaCl, 4.7KCl, 2.5 CaCl₂, 1.2 MgCl₂, 25 NaHCO₃, 5 Glucose, 0.4 Palmitate, 0.4 BSA,and 70 mU/l insulin. Perfusion pO₂>600 mmHg was maintained in theoxygenation chamber.

Evidence supporting the methods and products of the invention has beenestablished in an ex vivo model using hearts rapidly removed fromketamine/xylazine anesthetized 12-14 weeks old C57BL/6 mice weighingbetween 25-30 g that were retrograde perfused in a non-recirculatingmode, using an isovolumic perfusion system through the well-knownLangendorf System (FIG. 1). In this system isolated mouse hearts wereinitially perfused with a perfusion buffer containing KREB's solutioncontaining (in mM) the following: 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2MgCl₂, 25 NaHCO₃, 5 Glucose, 0.4 Palmitate, 0.4 BSA, and 70 mU/1insulin. Perfusion pO₂ at a pressure greater than 600 mmHg and a flowspeed of 2.5 ml/min was maintained in the oxygenation chamber. Theexperimental plan included an equilibration baseline period of 30 minnormoxic perfusion followed by 30 min global zero-flow ischemia (inducedischemia) and 60 min of reperfusion. The flow rate was 2.5 ml/min. Theperfusion apparatus was tightly temperature controlled for maintainingheart temperatures at 37+/−0.1° C. under all conditions. Baselinemeasurements were taken after 30 minutes of perfusion with the KREB'ssolution.

After the induced ischemia, control hearts were reperfused using thesame Kreb's buffer. Test hearts were reperfused in Kreb's buffer plus0.3% n-3 TG oil. The test omega-3 (n-3) triglyceride perfusion emulsionused to reperfuse test hearts was prepared using standard industrymethods for the production of the therapeutic emulsions in water. A 10%omega-3 triglyceride emulsion was obtained containing 10 ml n-3triglyceride (TG) that contained 48% fatty acids as EPA or DHA per 100ml water. This 10% TG emulsion was emulsified by egg yolk lecithin, 1.2g/100 ml. TG fatty acid (FA) composition (by weight) of the emulsionused in experiments 1-9 was determined by gas liquid chromatography andwas as follows for the n-3 emulsion: C14:0, 5.4%; C16:0, 14.8%; C16:1,8.4%; C18:0, 2.8%; C18:1, 12.7%; C18:2 (n-6), 2.6%; C18:3 (n-3), 0.8%;C20:1, 1.2%; C20:4 (n-6), 1.9%; C20:5 (EPA, n-3), 18.3%; C22:1, 0.2%;C22:4, 0.6%; C22:5, 2.6%; and C22:6 (DPA, n-3), 27.7. To make the testemulsion that was used to reperfuse the isolated hearts after theinduced ischemia, 300 mg 10% n-3 TG emulsion was diluted in 100 mlKreb's perfusion buffer to make a 0.3% n-3 TG perfusion emulsion,referred to in the summary of results as the test n-3 triglyceridereperfusion emulsion. The methods of the present invention use the n-3glyceride perfusion emulsions of the invention that have from about0.05% n-3 glyceride up to less than 7% n-3 glyceride.

Left Ventricular Developed Pressure (LVDP)

Left ventricular developed pressure (LVDP) was continuously monitoredusing a latex balloon placed on the left ventricle and connected to apressure transducer (Gould Laboratories; Pasadena, Calif.). Cardiacfunction measurements were recorded on a 2-channel ADI recorder. Acatheter inserted in the left ventricle of the isolated heart was usedto measure left ventricular developed pressure (LVDP) as well as cardiacrhythm in the ex vivo reperfusion experiments described.

Assay of Lactate Dehydrogenase (LDH)

Myocardial injury was assessed by measuring the release of lactatedehydrogenase (LDH) from the effluent in the ex vivo I/R system and fromblood samples in the in vivo LAD system, using the commerciallyavailable enzymatic kits (Pointe Scientific, INC, MI USA) as publishedearlier.^(1,2)

Western Blot Analysis

The tissue and cell protein concentration was determined using a DCProtein Assay kit (Bio-Rad). Equal amounts of protein were separated bySDS-PAGE (4-12% gradient gels), and proteins were loaded to anitrocellulose membrane (Invitrogen). After blocking nonspecific bindingwith the Odyssey blocking buffer (Li-Cor Biosciences), membranes wereincubated overnight at 4° C. with target primary antibodies (1:1,000dilution), according to the manufacturer's instructions. Successively,membranes were incubated with infrared labeled secondary antibodies for1 h at room temperature. The bound complex was visualized using theOdyssey Infrared Imaging System (Li-Cor; Lincoln, Nebr.). The imageswere analyzed using the Odyssey Application Software, version 1.2(Li-Cor) to obtain the integrated intensities.

Statistical Analysis

Data were expressed as the mean±SD. For assessing the difference betweenvalues, the Student's t test was used. A value of p<0.05 was consideredstatistically significant.

B. Results of Reperfusion of Isolated Hearts Ex Vivo with n-3 GlycerideEmulsion Reduces Reperfusion Injury after an Ischemic Event

After 1 hour of reperfusion following induced ischemia, the heart rateand LVDP were markedly decreased, and arrhythmias were observed bypressure recording in control hearts. FIG. 24. By contrast test heartsreperfused with the above-described n-3 triglyceride perfusion emulsionmaintained a normal heart rate, normal LVDP, and no or very fewarrhythmias. FIG. 25.

Specifically, after 1 hour of reperfusion with the n-3 triglycerideemulsion, there was over a 90% recovery of LVDP compared to only a 40%LVDP recovery in the control heart. FIG. 3. Heart rate recovery was alsoimproved with reperfusion in the n-3 triglyceride emulsion. In aseparate experiment, a 0.3% perfusion emulsion comprising the n-6 TGIntralipid™ was tested. FIG. 5.

Myocardial tissue injury was also assessed by measuring the release oflactate dehydrogenase (LDH) from the effluent in the ex vivo I/R systemand from blood samples in the in vivo left anterior descending coronaryartery ligation model system (LAD), using the commercially availableenzymatic kits (Pointe Scientific, INC, MI USA) as published earlier.Creatine kinase which is an additional marker of myocardial infarctionwas markedly decreased in isolated hearts that were reperfused in thetest n-3 triglyceride emulsion (FIG. 6). To determine if n-3 TG protectshearts by modulating changes in key signaling pathways linked to I/Rinjury, p-AKT, pGSK-3β, and Bcl-2 were probed in myocardial tissue bywestern blotting.

Since Bcl-2 interacts with Beclin-1, and influences autophagy, as shownin FIG. 14A-14C, n-3 TG treated hearts showed a significant reduction inBeclin-1 protein expression, with concomitant increase of Bcl-2 proteinexpression. Further, the level of the anti-apoptotic gene marker Bcl-2and expression of Bcl-2 mRNA were also increased in test n-3emulsion-reperfused hearts compared to control hearts (FIGS. 7A-7B).Next, hypoxia-inducible factor 1 (HIF-1), which is a key mediator ofadaptive responses to decreased oxygen availability in ischemia, wasexamined. HIF-1 protein expression (FIGS. 15A-15C) increased rapidlyafter ischemia. Another positive result showed that the level of proteinand mRNA for HIF-1, a marker for autophagy which may increase celldeath, were down-regulated after test hearts were reperfused with then-3 triglyceride emulsion, compared to controls (FIGS. 8A-8C). Anotherautophagic marker, Beclin 1, was also decreased in terms of proteinexpression and mRNA expression after reperfusion with buffer containingn-3 triglycerides (FIGS. 9A-8C). Finally, Western blot analysis showedthat PPAR gamma protein expression, associated with pro-inflammatoryresponses in heart, was markedly increased after reperfusion in controlhearts but decreased in test hearts that were reperfused in n-3 emulsion(FIG. 15A) (FIGS. 10A-10B).

The above findings tested in isolated, living, intact mammalian heart exvivo, show that including n-3 glycerides in a perfusion (or reperfusion)buffer for donor organs significantly preserved healthy function asindicated by a decrease in creatine kinase, and upregulation ofbeneficial proteins including the anti-apoptotic gene marker Bcl-2. N-3glycerides further resulted in downregulation of potentially harmfulproteins including HIF1 (a marker for autophagy which may increase celldeath), PPAR gamma (associated with some pro-inflammatory responses inheart), and beclin 1 (an autophagic marker).

Although only a 0.3% n-3 triglyceride perfusion emulsion was tested exvivo using the Langendorf system, the n-3 glyceride perfusion emulsionscan have higher amounts of up to but less than 7% n-3 oil, preferably0.5-1%, 1-2%, 2-5% or 5-7%, and they can have n-3 triglyceride, n-3diglyceride and combinations thereof. In previous studies for which someof the results are included in Examples 4 and 5, it was shown thatn-3-TG and n-3-DG-emulsions were very effective in reducing orpreventing reperfusion damage in vivo after hypoxia/ischemia. See alsoDeckelbaum, U.S. Ser. No. 11/558,568 and U.S. Ser. No. 13/336,290; U.S.Ser. Nos. 61/767,248, 12/441,795 (U.S. Pat. No. 8,410,181), Ser. Nos.13/783,779, 13/953,718, and 14/102,145. Thus it is expected that n-3glyceride perfusion emulsions of the present invention can be made usingthe n-3 DG and TG formulations that were administered in vivo.

Acute intervention (reperfusion) with 0.3% n-3 TG perfusion emulsion inthe ex vivo perfused heart (I/R model) after induced ischemia showedthat including n-3 TG significantly improved LVDP recovery after I/R(FIG. 12A), compared to control hearts. Reperfusion of the heart withKREB'S buffer+n-3 TG maintained normal rhythm and LVDP was nearlyrestored to 100% similar to pre-ischemia time. During reperfusionperiod, heart perfusates were collected to detect LDH release, asmarkers of ischemic injury. LDH release appeared significant differentbetween n-3 TG treated and control hearts, showing that acute n-3 TGtreatment exhibits a protective role (FIG. 12B).

To determine if n-3 TG protects hearts by modulating changes in keysignalling pathways linked to I/R injury, p-AKT, p-GSK-3β, and Bcl-2were probed after induced ischemia Reperfusion with 0.3% n-3triglyceride perfusion emulsion (0.3% n-3 TG) significantly increasedphosphorylation of AKT and GSK3β (FIGS. 13A-13C), and Bcl-2 proteinexpression (FIGS. 14A-14C), indicating that n-3 TG reduced apoptosis byactivating the PI3K-AKT-GSK3β signalling pathway and anti-apoptoticprotein Bcl-2. Since Bcl-2 interacts with Beclin-1^(3, 4) and influencesautophagy, as shown in FIG. 14A-14C, the expression of Beclin-1increased after ischemia/reperfusion condition; 0.3% n-3 TG treatedhearts showed a significant reduction in Beclin-1 protein expression,with concomitant increase of Bcl-2 protein expression as we mentionedabove. To establish the link between n-3 TG and PI3K/AKT and GSK3βpathways in I/R injury, hearts were treated with GSK-3β inhibitorSB216763 (3 μM) or Phosphatidylinositol 3-kinase (PI3K)/AKT inhibitorLY-294002 (10 μM); each of them was added at the beginning of thebaseline period and continued throughout ischemia and reperfusion. Thedoses of the inhibitors used in this study were based on publications inthe literature¹. LDH release was significantly reduced by 0.3% n-3 TG,and the protection thus afforded was abrogated by the PI3K/AKTinhibitor, LY-294002 (FIGS. 16A-16B). Treatment with SB-216763 plus 0.3%n-3 TG emulsion significantly inhibited LDH release compared to I/Rcontrol hearts (FIGS. 16A-16B).

Next, hypoxia-inducible factor 1 (HIF-1) was investigated, which is akey mediator of adaptive responses to decreased oxygen availability inischemia. HIF-1α protein expression (FIGS. 15A-15C) increased rapidlyafter ischemia. Inclusion of 0.3% n-3 TG in perfusion buffer duringreperfusion significantly inhibited the protein expression of HIF-1α.Previous studies showed that n-3 fatty acids, in contrast to saturatedfatty acids, are able to lower macrophages and arterial endotheliallipase and inflammatory markers and these effects are linked to PPAR-γ⁵.Accordingly, the potential association of PPAR-γ and n-3 TG acutetreatment in I/R condition was examined. Western blot analysis showedthat in n0.3% n-3 TG reperfused hearts protein expression of PPAR-γ wassignificantly lower compared to the control hearts (FIGS. 15A-15C).

In order to establish the link between PPAR-γ and n-3 TG effect, micewere treated with Rosiglitazone (6 mg/kg body weight, IP injection), acommon agonist of PPAR-γ, 30 min before I/R injury in the isolatedperfused hearts. These hearts were perfused with Krebs-Henseleit bufferwithout or with n-3 TG emulsion during reperfusion time. LDH release wassignificantly higher in Rosiglitazone plus n-3 TG treated hearts vsRosiglitazone treated hearts (FIGS. 16A-16B). These data indicate thatPPAR-γ reduction is linked to cardioprotection afforded by n-3 TG duringI/R.

Taken together, these results show that PI3K/AKT, GSK-3β and PPAR-γ arepathways modulating n-3 TG cardioprotection.

References Cited from Example 2

-   1. Ananthakrishnan, R., et al., Aldose reductase mediates myocardial    ischemia-reperfusion injury in part by opening mitochondrial    permeability transition pore. Am J Physiol Heart Circ Physiol, 2009.    296(2): p. H333-41.-   2. Hwang, Y. C., et al., Central role for aldose reductase pathway    in myocardial ischemic injury. FASEB J, 2004. 18(11): p. 1192-9.-   3. Griendling K K, and FitzGeral G A. (2003) Oxidative stress and    cardiovascular injury: part I: Basic mechanisms and in vivo    monitoring of ROS. Circulation, 108, 1912-1916.-   4. Marczin N, El-Habashi N, Hoare G S, Bundy R E, and    Yacoub M. (2003) Antioxidants in myocardial ischemia-reperfusion    injury: Therapeutic potential and basic mechanisms. Archives of    Biochemistry and Biophysics, 420, 222-236.-   5. Deckelbaum R J, Torrejon C. (2012) The omega-3 fatty acid    nutritional landscape: health benefits and sources. J Nutr.

Example 3: In Vivo Administration of Omega-3 Triglyceride DHA EmulsionsReduced Post Hypoxia-Ischemia Cardiac and Cerebral Reperfusion Injury

A. Materials and Methods

Lipid Emulsion

Four different types of lipid emulsions were tested. Omega-3 (n-3)triglyceride fish oil-based and omega-6 (n-6) soy oil-based emulsionswere commercially prepared intravenous phospholipid-stabilizedemulsions. The omega-3 triglyceride contained high concentrations of n-3fatty acids (FA) as described.^(1, 2) (See Table 1.) These omega-3triglyceride emulsions, referred to as “n-3 TG,” have 10% omega-3 fishoil (n-3) having less than 10% omega-6 oil by weight in grams per 100 mlof emulsion, wherein the omega-3 oil is >90% triglyceride (TG) by weightper total weight of the omega-3 oil, and in which up to about 30% % ofthe total acyl groups are DHA and up to about 28% % are EPA. The n-3 TGemulsions are also called “TG90-DHA30.” Other emulsions having pure(99%) DHA or pure (99%) EPA was also tested as described. For doses ofinjected n-3 TG emulsions, an amount was calculated to achieve anadministration containing 50% of the total TG-FA as DHA and EPA (Table1). Thus, 1 gm of TG emulsions is expressed as 0.5 gm n-3 TG.

The n-6 TG emulsions, referred to in the experiments in Example 3,comprise 20% omega-6 oil (n-6) by weight in grams per 100 ml ofemulsion, 0% DHA, 0% EPA and 55% TG from linoleic acid (Table 1). Thesen-6 TG emulsions were produced from soy bean oil rich in n-6 FA:linoleic acid constituting about 55% of total FA.

TABLE 1 Fatty Acid Composition of Lipid Emulsions¹ n-3 TG (g/100 ml)[herein “n-3 TG90-DHA30”] n-6 TG (g/100 ml) Source g/100 mL g/100 mLSoybean oil — 20 Fish oil 10 — Egg phosphatidylcholine 1.2 1.2 Glycerol2.5 2.25 FA (% of total FA by weight) % % Palmitic acid (C16:0) 2.5-10  7-14 Stearic acid (C18:0) 0.5-2   1.4-5.5 Oleic acid (C18: ln-9) >6-1319-30 Linoleic acid (C18:2n-6) 1-7 44-62 Arachidonic acid (C20: 4n-6)1-4 <0.5 α-linolenic acid (C18:3n-3) 2 >4-11 Eicosapentaenoic acid12.5-28.2 — (C22:6n-3) Docosahexaenoic acid (C22: 14.4-30.9 — 6n-3)¹Data provided by Fresenius Kabi AG; FA, Fatty acids.

Pure Tri-DHA (99% DHA) and Tri-EPA (99% EPA) emulsions were also made;they were VLDL-sized and laboratory-made with TG oil and egg yolkphospholipid using sonication and centrifugation procedures that areknown in the art.^(3, 4) Briefly, 200 mg of triglyceride-DHA oil>99% orTri-EPA oil>99% was mixed with a 5:1 weight ratio of egg yolkphosphatidylcholine (40 mg). The mixture was fully evaporated under N₂gas, and was further desiccated under vacuum overnight at 4° C. Thedried lipids were resuspended in 1 mL of lipoprotein-free buffer (LPB)(150 mmol/L NaCl, 0.5 ml of 0.1% glycerol and 0.24 mmol/L EDTA, pH 8.4,density 1.006 g/mL) at 60° C. with added sucrose (100 mg/l mL LPB) toremove excess phospholipid liposomes. The lipid emulsions were thensonicated for 1 hr at 50° C., 140 W under a stream of N₂ using a BransonSonifier model 450 (Branson Scientific, Melville, N.Y.). Aftersonication, the solution was dialyzed in LPB for 24 hr at 4° C. toremove sucrose. The final emulsions comprising VLDL-sized particles wereanalyzed for the amount of TG and PL by enzymatic procedure usingGPO-HMMPS, glycerol blanking method (Wako Chemicals USA, Inc., Richmond,Va.) and choline oxidase-DAOS method (Wako Chemicals USA, Inc.,Richmond, Va.). The TG: phospholipid mass ratio was 5.0±1.0:1 similar tothat of VLDL-sized particles. The emulsions were then stored under argonat 4° C. and were used within 2 weeks of preparation.

A specific example is of Tri-DHA was tested: “TG90-DHA30” that wasadministered in vivo following H/I to reduce reperfusion injury. Tri-DHAand Tri-EPA were purchased from Nu-Chek Prep, Inc. (Elysian, Minn.). Eggyolk phosphatidylcholine was obtained from Avanti Polar-Lipids, Inc.(Alabaster, Ala.).

B. Cardiac Reperfusion Injury is Reduced by Administration of n-3Triglycerides

In Vivo Left Anterior Descending Coronary Artery (LAD) Occlusion

In vivo murine model of ischemia-reperfusion injury: Prior to surgery,mice were anesthetized with isoflurane inhalation (4% induction followedby 1-2.5% maintenance). Subsequent to anaesthesia, mice were orallyintubated with polyethylene-60 (PE-60) tubing, connected to a mouseventilator (MiniVent Type 845, Hugo-Sachs Elektronik) set at a tidalvolume of 240 μL and a rate of 110 breaths per minute, and supplementedwith oxygen. Body temperature was maintained at 37° C. A mediansternotomy was performed, and the proximal left coronary artery (LAD)was visualized and ligated with 7-0 silk suture mounted on a taperedneedle (BV-1, Ethicon). After 30 min of ischemia, the prolene suture wascut and the LAD blood flow was restored. Immediately after,intraperitoneal (IP) injection of n-3 TG emulsion (1.5 g/kg body weight)was performed and the second injection was done after 60 min ofreperfusion. Control animals received IP injection of saline solutionfollowing the same time course. The chest wall was closed, and mice weretreated with buprenorphine and allowed to recover in atemperature-controlled area^(4, 5).

Echocardiogram

In vivo transthoracic echocardiography was performed using a VisualSonics Vevo 2100 ultrasound biomicroscopy system. This high-frequency(40 MHz) ultrasound system has an axial resolution of ˜30-40 microns anda temporal resolution of >100 Hz. Baseline echocardiography images wasobtained prior to myocardial ischemia and post-ischemic images wereobtained after 48 hours of reperfusion. The mice were lightlyanesthetized with isoflurane (1.5-2.0 L/min) in 100% O₂ and in vivotransthoracic echocardiography of the left ventricle (LV) using a MS-40038-MHz microscan transducer was used to obtain high resolution twodimensional mode images. Images were used to measure LV end-diastolicdiameter (LVEDD), LV end-systolic diameter (LVESD), LV ejection fraction(EF) and LV fractional shortening (FS) as published earlier.^(4, 5)

Infarct Size Measurement

Myocardial infarct size determination: At 48 h of reperfusion mice werere-anesthetized, intubated, and ventilated using a mouse ventilator. Acatheter (PE-10 tubing) was placed in the common carotid artery to allowfor Evans blue dye injection. A median sternotomy was performed and theLAD was re-ligated in the same location as before. Evans blue dye (1.25ml of a 7.0% solution) was injected via the carotid artery catheter intothe heart to delineate the non-ischemic zone from the ischemic zone. Theheart was then rapidly excised and fixed in 1.5% agarose. After the gelsolidified, the heart was sectioned perpendicular to the long axis in1-mm sections using a tissue chopper. The 1-mm sections was placed inindividual wells of a six-well cell culture plate and counterstainedwith 1% TTC for 4 min at 37° C. to demarcate the nonviable myocardium.Each of the 1 mm thick myocardial slices was imaged and weighed. Imageswere captured using a Q-Capture digital camera connected to a computer.Images were analysed using computer-assisted planimetry with NIH Image1.63 software to measure the areas of infarction and total riskarea.^(4, 5)

C. Results of 3 TG Administration Reduces Infarct Size and ImprovedCardiac Function in LAD Model.

To test the effect of acute n-3 TG administration in myocardial ischemicinjury, mice were subjected to 30 min of ischemia induced by LADocclusion; coronary flow was then restored and myocardial functionalrecovery during reperfusion was assessed. IP injection of n-3 TGemulsion was administered immediately after ischemia at the onset ofreperfusion and at 60 min into reperfusion. At the end of 48 h ofreperfusion, sections of heart were stained with TTC to quantify theextent of I/R damage in both groups. FIG. 11A shows quantification ofthe infarct area in mice hearts from saline treated compared to n-3 TGtreated group. Myocardial infarct size was significantly reduced(p<0.05) in n-3 TG emulsion treated mice (vs saline treated mice). Thetotal area at risk was similar for both groups. Plasma LDH release, akey marker of myocardial injury, was significantly reduced in n-3 TGtreated mice. FIG. 11B. These data indicate that acute treatment of n-3TG during reperfusion markedly reduces injury due to myocardialinfarction in mice.

Echocardiography assessment showed substantial differences in fractionalshortening (% FS) between control and n-3 TG treated mice. A significantrecovery of FS was observed in n-3 TG treated group vs saline treatedcontrols (p<0.01) (FIG. 11C). These data along with infarct size changesand LDH levels reduction reveal that acute n-3 TG treatment protectsmice from myocardial ischemia-reperfusion injury and improves heartfunction.

References Cited in Example 3

-   1. Oliveira F L, Rumsey S C, Schlotzer E, Hansen I, arpentier Y A,    et al. (1997) Triglyceride hydrolysis of soy oil vs fish oil    emulsions. JPEN J Parenter Enteral Nutr 21: 224-229.-   2. Qi K, Seo T, Al-Haideri M, Worgall T S, Vogel T, Carpentier Y A,    Deckelbaum R J. (2002) Omega-3 triglycerides modify blood clearance    and tissue targeting pathways of lipid emulsions. Biochemistry.    41(9): 3119-27.-   3. Qi K, Al-Haideri M, Seo T, Carpentier Y A, Deckelbaum R J (2003)    Effects of particle size on blood clearance and tissue update of    lipid emulsions with different triglyceride compositions. JPEN J    Parenter Enteral Nutr 27: 58-64.-   4. Schwiegelshohn B, Presley J F, Gorecki M, Vogel T, Carpentier Y    A, et al. (1995) Effects of apoprotein E on intracellular metabolism    of model triglyceride-rich particles are distinct from effects on    cell particle update. J Biol Chem 270: 1761-1769.-   5. Ananthakrishnan, R., et al., Aldose reductase mediates myocardial    ischemia-reperfusion injury in part by opening mitochondrial    permeability transition pore. Am J Physiol Heart Circ Physiol, 2009.    296(2): p. H333-41.-   6. Hwang, Y. C., et al., Central role for aldose reductase pathway    in myocardial ischemic injury. FASEB J, 2004. 18 (11): p. 1192-9.

Example 4: In Vivo Administration of Triglycerides with High DHA Contentafter Cerebral Hypoxia-Ischemia Reduces Reperfusion Injury

A. Materials and Methods

Amounts of n-3 TG in the Lipid Emulsions are as Described Above.

Induction of Unilateral Cerebral H/I

Three-day-old C57BL/6J neonatal mice of both genders were purchased fromJackson Laboratories (Bar Harbor, Me.) with their birth mother. TheRice-Vannucci model of H/I was used and modified to p10 neonatal mice.⁵Briefly, on postnatal day 10 H/I was induced by the ligation of theright common carotid artery, which was further cauterized and cut underisoflurane anesthesia. The investigator was blinded to the lipidemulsion treatment during the surgery and after the surgery. The entiresurgical procedure was completed within 5 min for each mouse. Pups werethen allowed to recover with their dams for 1.5 hr. Surroundingtemperature during experiments was kept at 28° C. Mice were then exposedto systemic hypoxia for 15 min in a hypoxic chamber in a neonatalisolette (humidified 8% oxygen/nitrogen, Tech Air Inc., White Plains,N.Y.).⁵ The ambient temperature inside the chamber during hypoxia wasstabilized at 37±0.3° C. To minimize a temperature-related variabilityin the extent of the brain damage, during the initial 15 hr ofreperfusion mice were kept in an isolette at the ambient temperature of32° C.

Quantification of Brain Infarction

After 24 hr of reperfusion, the animals were sacrificed by decapitationand brains were immediately harvested. 1-mm coronal slices were cut byusing a brain slicer matrix. Slices were then immersed in a PBS solutioncontaining 2% triphenyl-tetrazolium chloride (TTC) at 37° C. for 25 min.TTC is taken up into living mitochondria, which converts it to a redcolor.⁶ Thus, viable tissue stains brick-red, and nonviable (infarcted)tissue can be identified by the absence of staining (white). Using AdobePhotoshop and NIH Image J imaging applications, planar areas ofinfarction on serial sections were summed to obtain the volume (mm³) ofinfarcted tissue, which was divided by the total(infarcted+non-infarcted) volume of the hemisphere ipsilateral tocarotid artery ligation, and expressed as a percentage of total volume.

Experimental Groups

H/I brain injury was induced in different groups of animals, whichreceived specific treatments before and after H/I injury. Animalsfollowed different treatment protocols.

Protocol 1: Pre-H/I Treatment of n-3 TG (Containing Both DHA and EPA) orn-6 TG Emulsions.

Two doses of n-3 TG or n-6 TG emulsions or vehicle (saline, equalvolumes/kg) were administered to non-fasting rodents at a fixed dose of3 mg of n-3 or n-6 TG-FA per mouse for each injection (equivalent to amaximum of 1.5 g of total TG/kg; p10 mice weighed 4-6 gm for theseexperiments). The first dose was i.p. administered immediately aftersurgery, and the second immediately at the end of the 15 min hypoxicperiod. Volumes injected for TG emulsions and saline were always equal.

Since n-3 emulsions contain low concentrations of alpha-tocopherol as ananti-oxidant agent, in separate experiments an equivalent dose of purealpha-tocopherol to match the content of n-3 emulsion content (0.8 g/L)was given to neonatal mice by i.p. injection of alpha-tocopherol (VitalE®, Intervet, Schering Plough) at a dose of 5 mg alpha-tocopherol/kgbody weight, the amount contained in each i.p. injection of the n-3 TGemulsions.

Protocol 2: Post-H/I Treatment of n-3 TG (Containing Both DHA and EPA).

Two doses of the commercially available n-3 TG emulsion or saline werei.p. injected into non-fasting rodents at 0.75 g of n-3 TG/kg bodyweight for each dose (equivalent to 1.5 g of total TG/kg). The firstdose was administered immediately after 15-min hypoxia, and the secondat 1 hr after start of the reperfusion period.

Protocol 3: Dose Response, Timing and Specificity of n-3 TG.

Two types of n-3 containing lipid emulsions either Tri-DHA or Tri-EPA(0.1 g n-3 TG/kg or 0.375 g n-3 TG/kg body weight for each dose) wereadministered twice to non-fasting rodents according to the amount of DHAand EPA in the mixed n-3 TG emulsions. See Table 1. The first dose wasinitially administered immediately after 15-min hypoxia, and the secondafter 1 hr of reperfusion. Then in different sets of experiments, theefficacy of Tri-DHA emulsions was determined, with the initial injectionadministered at four-time points (0 hr, or at 1-hr, 2-hr or 4-hr afterH/I), 0.375 g n-3 TG/kg body weight for each dose. For the immediatetreatment of 0 hr, the first dose was injected immediately after 15-minhypoxia, with a second injection after 1 hr of reperfusion, whereas inthe “delayed” treatments, the first dose was given after the 1^(st) or2^(nd) or 4^(th) hr of reperfusion and a second dose was administered 1hr after the 1^(st) dose.

Measurement of Blood TG and Glucose Levels

Blood samples for blood TG were directly taken from left ventricle ofhearts under isoflurane inhalation from a separate cohort ofnon-fasting, 10-day-old mice. Samples were taken over a 5 hr periodafter a single i.p. injection of either 0.75 g n-3 TG/kg commerciallyavailable n-3 rich TG (DHA and EPA) emulsions or saline. Total plasma TGwas enzymatically measured by GPO-HMMPS, glycerol blanking method (WakoChemicals USA, Inc., Richmond, Va.). For glucose levels, blood sampleswere taken from mouse tails from a separate cohort of non-fasting10-day-old mice. Samples were taken at two time points from each mouse.The first sample was taken at time zero before surgery and TG injection,and the second at about 10 min after H/I and TG injection (approximately100 min after surgery as described under the Unilateral Cerebral H/Iprotocol above). Blood glucose levels were electrochemically measured inmg/dL by a glucose meter (OneTouch Ultra, LifeScan, Inc., Milpitas,Calif.).

Measurement of Cerebral Blood Flow (CBF) by Laser Doppler Flowmetry(LDF)

In a cohort of neonatal C57BL/6J mice pups subjected to carotid arteryligation and recovery as described above, relative CBF was measuredduring hypoxia in ipsilateral (right) hemispheres using a laser Dopplerflow meter (Periflux 5000). In these mice, in preparation for CBFmeasurement the scalp was dissected under isoflurane anesthesia andDoppler probes were attached to the skull (2 mm posterior and 2 mmlateral to the bregma) using fiber optic extensions. Only localanesthesia (1% lidocaine) was used postoperatively. Mice were thenplaced into a hypoxia chamber (8% O₂/92% N₂). Changes in CBF in responseto hypoxia were recorded for 20 min and expressed as percentage of thepre-hypoxia level for n-3 treated and saline treated neonatal mice.

Measurement of Bleeding Time after n-3 TG Injection

Bleeding times were measured in mice after severing a 3-mm segment ofthe tail.⁷ Two doses of saline were administered vs. n-3 TG in a similartime frame as the original protocol: an initial injection followed by asecond injection at 2 hr later. Bleeding times were measured at 45 minafter the second dose. The amputated tail was immersed in 0.9% isotonicsaline at 37° C., and the time required for the stream of blood to stopwas defined as the bleeding time. If no cessation of bleeding occurredafter 10 min, the tail was cauterized and 600 s was recorded as thebleeding time.

Long-Term Assessment of Brain Tissue Death

A long-term assessment of cerebral injury was performed at 8 wk afterneonatal H/I insult. This cohort of mice at p10 underwent unilateral H/Ifollowed by post H/I injections with either 0.375 g Tri-DHA/kg (n=6) orsaline (n=5) as described above. At 8 wk after H/I, mice were sacrificedby decapitation. Brains were removed, and embedded in TissueTek-OTC-compound (Sakura Fineteck, Torrance, Calif.) with subsequentsnap freezing in dry ice-chilled isopentane (−30° C.), and stored at−80° C. For analysis, coronal sections (10 μm every 500 μm) were cutserially in a Leica cryostat and mounted on Superfrost slides (ThermoScientific, Illinois). Sections were processed for Nissl staining byusing Cresyl Violet Acetate (Sigma-Aldrich, St. Louis, Mo.). Using AdobePhotoshop and NIH Image J imaging applications, 9 sections from eachbrain containing both the right and left hemispheres were traced forbrain tissue area. As previously described⁸ the area of left control orcontralateral hemisphere which had not had injury was given a value in100% for each animal. The brain area remaining in the right injuredipsilateral hemisphere was then compared to the left hemisphere, and thedifference was taken as the percent right brain tissue loss, for eachanimal.

Statistical Analysis

Data are presented as mean±SEM. Plasma TG levels were compared at eachtime point after i.p. injection of n-3 TG emulsion. Student t tests wereused for 2-group comparisons. 1-way ANOVA, followed by Bonferroniprocedure for post hoc analysis to correct for multiple comparisons, wasused to compare the differences among the emulsions on the infarct areasacross coronal sections. Statistical significance, which was analyzed byusing SPSS software 16.0 (SPSS Inc., Chicago, Ill.), was determined atp<0.05.

B. Results of In Vivo Administration of n-3 Tri-DHA after CerebralHypoxic-Ischemia Reduced Reperfusion Damage

Effects of On-3 Tri-DHA on Blood Triglyceride, Blood Glucose Levels,Bleeding Time and Cerebral Blood Flow after H/L

TG levels of saline-injected mice remained constant over 5 hr after i.p.injection reflecting normal blood TG levels in neonates. Further therewas no difference in blood glucose levels treated with n-3 TG comparedto controls (FIG. 17). After H/I insult, blood glucose levels decreasedsimilarly, about 30% or more, in all groups (p<0.05). There was nodifference in capillary bleeding times in n-3 treated mice (437±82 sec)as compared to saline controls (418±90 sec). Further, very similar bloodflow levels were maintained in neonatal H/I mice whether they weresaline treated or n-3 treated in this model.

n-3 TG but not n-6 TG Protected the Brain Against H/I Injury

Coronal sections of brains were stained with TTC to quantify the extentof post H/I brain injury and the effect of n-3 TG [and n-6 THG]injection. FIG. 3A shows representative images of neonatal mouse brainfrom saline treated, n-6 TG-emulsion-treated and n-3 TG90-DHA30emulsion-treated mice with pre-and-post injection after H/I,respectively. In all H/I animals, tissue death was localized to theright hemisphere (ipsilateral to ligation) as illustrated by the whiteareas in the upper panels of FIG. 18A. The image in the lower panels,demonstrated tracings of the infarcted areas for quantifying infarctvolume using NIH Image J. The brains from saline treated animalsexhibited a consistent pannecrotic lesion involving both cortical andsubcortical regions ipsilateral to the ligation. In the majority of theanimals the neuroprotection after n-3 TG injection was most marked inthe subcortical area, whereas saline treated mice had large cortical andsubcortical infarcts.

Infarct volume was substantially decreased in n-3 TG treated mice (n=28)compared to saline treated littermates (control) (n=27), 19.9±4.4% vs.35.1±5.1%, respectively (p=0.02). See FIG. 18B. There was a significantincrease in infarct volume with n-6 TG emulsion injection compared tosaline control (p=0.03) and the n-3 TG groups (p<0.01).

Because alpha-tocopherol is a component of the TG emulsions (present inlow concentrations to prevent FA oxidation) TTC staining was used tocompare the extent of cerebral H/I injury in alpha-tocopherol treatedand saline treated neonatal mice. There was no significant difference ininfarct volume between brains in alpha-tocopherol injected mice comparedto saline treated mice (data not shown).

It was then determined if n-3 TG90-DHA30 were effective if injected onlyafter H/I (without injection prior to H/I. See FIG. 18C. Similarly, thesmaller n-3 TG90-DHA30 associated lesions were mainly subcortical (datanot shown). Compared to saline controls in the immediate post-H/Itreatment the total infarct area was significantly reduced almost 50% inthe n-3 TG90-DHA30 post H/I-treated group.

DHA but not EPA is Neuroprotective after H/I

To determine possible differences in neuroprotection of EPA vs. DHA, theextent of brain injury was studied using the post-H/I treatment protocoladministering 99% DHA (n-3 99% Tri-DHA) vs. 99% EPA (n-3 99% Tri-EPA)emulsions in two dosages (0.1 g TG/kg vs. 0.375 g TG/kg). No statisticaldifferences in brain infarct volume between the two doses 0.1 g TG/kgand 0.375 g TG/kg in the 99% Tri-DHA-treated groups were observed.However, compared to saline control, total infarct size was reduced by amean of 48% and 55% by treatment with 0.1 and 0.375 g 99% Tri-DHA/kg,respectively. See FIG. 19A-19B. Neuroprotection was not observed with99% Tri-EPA injection at either of the two doses compared with salinetreatment.

To better approximate realistic timelines for neuroprotection afterstroke for humans delayed treatment protocols were performed in aneffort to study the therapeutic window of Tri-DHA emulsions. Noprotective effect from TG90-DHA30 administration after a 4-hr delay intreatment was noted when compared with saline group. However, TG90-DHA30administered at 0 hr immediately post H/I, and then delayed 1-hr and2-hr post stroke showed similar reduced (˜50%) brain infarct volumescompared to saline treated animals. FIG. 20 shows right brain tissueloss in saline- vs Tri-DHA-treated animals. This substantial protectionoccurred mainly in subcortical areas similar to the findings describedabove.

Long-Term Neuroprotection

Coronal brain sections of adult mice were processed for Nissl staining(FIG. 6) to examine the effects of H/I and TG90-DHA30 treatment on brainand neuronal cell loss for long-term outcome at 8 wk after H/I insult.As compared to the left control (contralateral hemisphere), the injuredareas of the right hemisphere display gross neuronal cell loss. As shownin FIG. 21, brain tissue loss was markedly increased by 1.67 fold in theright hemisphere of saline-treated mice (n=5) as compared to TG90-DHA30treated mice (n=6), 25.0±2.4% vs. 15.0±2.5%, respectively (p=0.02).Thus, neuroprotection after injury and TG90-DHA30 injection that areobserved 24 hr after H/I can be demonstrated histologically almost 2months after the initial stroke insult.

References Cited from Example 4

-   1. Oliveira F L, Rumsey S C, Schlotzer E, Hansen I, Carpentier Y A,    et al. (1997) Triglyceride hydrolysis of soy oil vs fish oil    emulsions. JPEN J Parenter Enteral Nutr 21: 224-229.-   2. Qi K, Seo T, Al-Haideri M, Worgall T S, Vogel T, et al. (2002)    Omega-3 triglycerides modify blood clearance and tissue targeting    pathways of lipid emulsions. Biochemistry 41: 3119-3127.-   3. Qi K, Al-Haideri M, Seo T, Carpentier Y A, Deckelbaum R J (2003)    Effects of particle size on blood clearance and tissue uptake of    lipid emulsions with different triglyceride compositions. JPEN J    Parenter Enteral Nutr 27: 58-64.-   4. Schwiegelshohn B, Presley J F, Gorecki M, Vogel T, Carpentier Y    A, et al. (1995) Effects of apoprotein E on intracellular metabolism    of model triglyceride-rich particles are distinct from effects on    cell particle uptake. J Biol Chem 270: 1761-1769.-   5. Ten V S, Bradley-Moore M, Gingrich J A, Stark R I, Pinsky D    J (2003) Brain injury and neurofunctional deficit in neonatal mice    with hypoxic-ischemic encephalopathy. Behav Brain Res 145: 209-219.-   6. Liszczak T M, Hedley-Whyte E T, Adams J F, Han D H, Kolluri V S,    et al. (1984) Limitations of tetrazolium salts in delineating    infarcted brain. Acta Neuropathol 65: 150-157.-   7. Denis C, Methia N, Frenette P S, Rayburn H, Ullman-Cullere M, et    al. (1998) A mouse model of severe von Willebrand disease: defects    in hemostasis and thrombosis. Proc Natl Acad Sci USA 95: 9524-9529.-   8. Seo T, Blaner W S, Deckelbaum R J (2005) Omega-3 fatty acids:    molecular approaches to optimal biological outcomes. Curr Opin    Lipidol 16: 11-18.-   9. Bruno A, Biller J, Adams H P, Jr., Clarke W R, Woolson R F, et    al. (1999) Acute blood glucose level and outcome from ischemic    stroke. Trial of ORG 10172 in Acute Stroke Treatment (TOAST)    Investigators. Neurology 52: 280-284.

Example 5: In Vivo Cerebral Hypoxia/Ischemia Treatment with 20% n-3 TGEmulsion Reduces Reperfusion Damage

A. 60 Min. Hypoxia in Mice

Postnatal day 19-21 Wistar rats of both genders were subjected tounilateral (right) carotid artery ligation. See Rice, J. E., 3rd, R. C.Vannucci, et al. (1981), “The influence of immaturity onhypoxic-ischemic brain damage in the rat,” Ann Neurol 9(2): 131-41 andVannucci, S. J., L. B. Seaman, et al. (1996), “Effects ofhypoxia-ischemia on GLUT1 and GLUT3 glucose transporters in immature ratbrain,” Journal of Cerebral Blood Flow & Metabolism 16(1): 77-81.

Immediately after ligation, six rats were given 50 mg 20% omega-3lipid-based emulsion (0.25 cc)(a 20% long chain omega-3triglyceride-based formula having >45% of total omega-3 fatty acid aseicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) viaorogastric feeding tube, and six control rats were given 0.25 cc water,both enterally. The 20% omega-3 lipid-based emulsion was made placing 20gm of omega-3 triglyceride in 100 ml of water, and emulsifying with 1.2gm of egg yolk lecithin. Rats were allowed to recover for 2 hours andthen they underwent hypoxia-ischemia for 60 minutes of 8% oxygen at aconstant temperature. The six pre-treated rats were given another doseof 50 mg omega-3 lipid-based emulsion immediately after thehypoxia-ischemia and control rats were given 0.25 cc water. All ratswere euthanized at 72 hours of reperfusion. The brains were removed andcut into 2 mm sections and stained with 2, 3, 5,Triphenyl-2H-tetrazolium chloride (TTC). TTC is a vital die that stainscells red that have respiring mitochondria. Dead tissue (infarct)appears white. The sections were scored as follows:

-   -   0—no evidence of edema or cell death    -   1—edema without cell death    -   2—edema with minimal cell death    -   3—edema with significant cell death

All rats survived 60 minutes of hypoxia-ischemia. Six of the six controlrats had edema and/or cell death with a mean score of 2+/−0.83 (standarddeviation), while two of the six treated rats had damage with a meanscore of 0.42+/−0.62 (p<0.005).

B. 65 Min Hypoxia

Postnatal day 19-21 Wistar rats of both genders were subjected tounilateral (right) carotid artery ligation. Immediately after ligation,six rats were given 50 mg 20% omega-3 lipid-based emulsion (0.25 cc)(20%omega-3 fatty acid based formula having ≥40% of total omega-3 fatty acidas EPA and DHA) via orogastric feeding tube and six control rats weregiven 0.25 cc water, both enterally. The emulsion was made as describedin Example 1. The rats recovered for two hours, and then underwenthypoxia-ischemia for 65 minutes of 8% oxygen at a constant temperature.The six pre-treated rats were given another dose of 50 mg omega-3lipid-based emulsion immediately after the hypoxia-ischemia and controlrats were given 0.25 cc water. All rats were euthanized at 72 hours ofreperfusion. The brains were removed and cut into 2 mm sections andstained with 2, 3, 5, Triphenyl-2H-tetrazolium chloride (TTC). Thesections were scored as above.

The 65 or 60 minutes of hypoxia-ischemia produced damage in all rats.Four of the six control rats survived with a mean score of 2.75+/−0.50,while five of the six treated rats survived with a mean score of1.70+1=0.76 (p<0.05).

C. Treatment of Rats with Omega-3 Triglyceride Lipid Emulsion Prior to60 Minutes of Hypoxia

Postnatal day 19-21 Wistar rats were subjected to unilateral (right)carotid artery. Immediately after ligation, six rats were given 50 mg ofa 20% omega-3 lipid-based emulsion (0.25 cc), and six control rats weregiven 0.25 cc water, both enterally. The emulsion was as described abovein Example 1. Rats were allowed to recover for two hours, and thenunderwent hypoxia-ischemia for 60 minutes of 8% oxygen at a constanttemperature. The six pre-treated rats were given another dose of 50 mgomega-3 triglyceride lipid emulsion immediately after thehypoxia/ischemia and control rats were given 0.25 cc water. At 72 hoursof reperfusion, the rats were euthanized and their brains removed, cutinto 2 mm sections and stained with 2,3,5 triphenyl-2H-tetrazoliumchloride (TTC). The damage in each animal was then given a score from 0(no damage) to 4 (>60% ipsilateral hemisphere infarcted). All of thevehicle-treated animals suffered brain damage, with a mean damage scoreof 2.00+0.89; the omega-3 triglyceride lipid emulsion-treated rats weresignificantly less damaged, having a mean damage score 0.33+0.52,p<0.05. The size of brain infarcts was determined by TTC staining. Notereceived enteral administration of TG emulsion.

These results show that when omega-3 triglycerides were administeredeither immediately before and/or after hypoxia-ischemia they confer asignificant neuroprotection. Very similar results were obtained when theomega-3 triglycerides were injected parenterally.

D. Treatment Following Hypoxic Ischemia

Post-natal day 19-21 rat pups were subjected to unilateral carotidartery ligation and 60 minutes of hypoxic ischemia, according to thepreviously described protocol. On four separate occasions, rats weretreated by parenteral injection of omega-3 lipid-based emulsion (100 mg)immediately after the insult, and again at four hours after the insult.The emulsion was as described above in Example 1. Brain damage wasevaluated by TTC staining at 72 hours of reperfusion. In each instance,administration of the omega-3 lipid-based oil emulsion provided greaterthan 50% protection, i.e. reduction of tissue damage.

A total of 14 control subjects (saline-treated) and 21 treated subjects(omega-3 lipid-based emulsion treated) were included in the experiment.Mean damage scores were: 1.93±0.22 (SEM), control, 0.78±0.16emulsion-treated; p<0.0001 by two-tailed test. Thus, in addition to thesignificance of the overall protection, it can be seen that 40% of thetreated animals were 100% protected (no damage at all, compared to 1/14untreated; 40% suffered only mild damage, compared to 1/14 mildlydamaged untreated animals. These results indicate that treatmentfollowing hypoxic-ischemia provides a neuroprotective benefit asindicated by a reduction of tissue damage.

Preliminary experiments conducted in the adult mouse show a comparablelevel of neuroprotection from hypoxic-ischemic damage. (Data not shown.)

Fatty acyl composition analyses of brain lipids (by gas liquidchromatography) after hypoxia/ischemia showed no relative differencesbetween infarcted brain versus non infarcted brain indicating thateffects of acute administration of omega-3 emulsions were not dependenton fatty acid compositional changes in brain membranes. In the infarctedareas, however, absolute concentrations of all fatty acids fell tosimilar degrees by about 15% (μg fatty acid per gram wet brain)indicating brain edema. This decrease did not occur with administrationof omega-3 emulsions indicating that these omega-3 fatty acids preventedthe brain edema as well as infarction.

The invention has been described with reference to specific embodiments.It will, however, be evident that various modifications and changes maybe made thereto without departing from the broader spirit and scope ofthe invention. The specification and drawings are, accordingly, to beregarded in an illustrative rather than a restrictive sense. Theinvention is illustrated herein by the experiments described above andby the following examples, which should not be construed as limiting.The contents of all references, pending patent applications andpublished patents, cited throughout this application are herebyexpressly incorporated by reference. Although specific terms areemployed, they are used as in the art unless otherwise indicated.

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What is claimed is:
 1. An omega-3 oil in water emulsion application forperfusion of an organ or tissue ex vivo, wherein the emulsion comprises(a) a perfusion buffer suitable for organ or tissue preservation andtransplantation, (b) from 0.05% to 0.5% of an omega-3 oil by weight ingrams per 100 ml of perfusion buffer, wherein the omega-3 oil (i)comprises from about 10% to about 99% omega-3 diglyceride, omega-3triglyceride or combinations thereof by weight per total weight of theomega-3 oil, and at least about 20% to about 99% of the total acylgroups of the diglycerides and triglycerides comprise EPA or DHA orcombinations thereof, and (ii) comprises less than 10% omega-6 fattyacids by weight per total weight of the omega-3 oil, (c) less than 10%omega-6 oil by weight in grams per 100 ml of perfusion buffer, and (d)the mean diameter of lipid droplets in the emulsion is from about 100 nmto less than about 5 microns.
 2. The omega-3 oil in water emulsion ofclaim 1, wherein the emulsion comprises from about 0.05% to about 0.3%in grams per 100 ml of perfusion buffer.
 3. The omega-3 oil in wateremulsion of claim 1, wherein from about 20% to about 40% of the totalacyl groups of the omega-3 diglycerides and triglycerides comprise EPA,DHA or a combination thereof.
 4. The omega-3 oil in water emulsion ofclaim 1, wherein from about 40% to about 60% of the total acyl groups ofthe omega-3 diglycerides and triglycerides comprise EPA, DHA or acombination thereof.
 5. The omega-3 oil in water emulsion of claim 1,from about 60% to about 80% of the total acyl groups of the omega-3diglycerides and triglycerides comprise EPA, DHA or a combinationthereof.
 6. The omega-3 oil in water emulsion of claim 1, from about 80%to about 99% of the total acyl groups at the omega-3 diglycerides andtriglycerides comprise EPA, DHA or a combination thereof.
 7. The omega-3oil in water emulsion of claim 1, wherein the lipid droplets are lessthan about 1 micron in diameter.
 8. The omega-3 oil in water emulsion ofclaim 1, wherein the lipid droplets are from about 100 nm to about 500nm in diameter.
 9. The omega-3 oil in water emulsion of claim 1, whereinthe perfusion buffer comprises an aqueous solution at a physiologic pH.10. An omega-3 oil in water emulsion for cold storage of an organ ortissue ex vivo, wherein the emulsion comprises (a) storage solutionsuitable for organ or tissue preservation and transplantation, (b) fromabout 0.05% less than 0.5% of an omega-3 oil by weight in grams per 100ml of the storage solution suitable for organ or tissue preservation andtransplantation, wherein the omega-3 oil (i) comprises from about 10% toabout 99% omega-3 diglyceride, omega-3 triglyceride or combinationsthereof by weight per total weight of the omega-3 oil, and at leastabout 20% to about 99% of the total acyl groups of the diglycerides andtriglycerides comprise EPA or DHA, and (ii) comprises less than 10%omega-6 fatty acids by weight per total weight of the omega-3 oil, (c)less than 10% omega-6 oil by weight in grams per 100 ml of perfusionbuffer, and (d) the mean diameter of lipid droplets in the emulsion isfrom about 100 nm to less than about 5 microns.